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Sample records for a1 proteases evidence

  1. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases.

    Batten, Margaret R; Senior, Bernard W; Kilian, Mogens; Woof, Jenny M

    2003-03-01

    The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors. PMID:12595464

  2. Immunoglobulin A1 protease activity in Gemella haemolysans

    Lomholt, JA; Kilian, Mogens

    2000-01-01

    The purpose of this study was to determine the occurrence and nature of immunoglobulin A1 (IgA1) protease activity in members of the genus Gemella and related taxa. Among a total of 22 Gemella strains belonging to the four species Gemella haemolysans, Gemella morbillorum, Gemella sanguinis, and...

  3. Bacterial retropepsin-like proteases : the evidence from Legionella pneumophila

    Teixeira, Paulo Alexandre Gonçalves

    2013-01-01

    A familia A2 de proteases aspárticas é constituida maioritariamente por proteases encontradas em retrovírus – as retropepsinas. As teorias evolutivas inerentes a estas proteases normalmente referem que estarão relacionadas com proteases do tipo pepsina pertencentes à familia A1 de proteases aspárticas. Pela primeira teoria (geralmente a mais aceite), durante a infeção de uma célula eucariota por um retrovírus, o gene da retropepsina terá sofrido duplicação e fusão dando orig...

  4. Antigenic relationships among immunoglobulin A1 proteases from Haemophilus, Neisseria, and Streptococcus species.

    Lomholt, H.; Kilian, M

    1994-01-01

    To investigate the antigenic variation and relationships of immunoglobulin A1 (IgA1) proteases among different species and genera, we examined a comprehensive collection of serine type and metallo-type IgA1 proteases and corresponding antisera in enzyme neutralization assays. Sharing of neutralizing epitopes of metallo-type IgA1 proteases from Streptococcus pneumoniae, Streptococcus sanguis, Streptococcus mitis, and Streptococcus oralis and of serine type IgA1 proteases from Haemophilus and p...

  5. Purification and characterization of an immunoglobulin A1 protease from Bacteroides melaninogenicus.

    Mortensen, S B; Kilian, M

    1984-01-01

    Attention has recently been focused on bacterial proteases with the capacity to cleave immunoglobulin A (IgA proteases) as possible pathogenic factors in bacterial meningitis, gonorrhoea, and destructive periodontal disease. Here, we describe a method for the rapid purification of a specific IgA1 protease from Bacteroides melaninogenicus. The IgA1 protease was purified 6,172-fold with a yield of 9% by ammonium sulfate precipitation, DEAE-ion exchange chromatography, and separation on a prepar...

  6. Characterizing proteases in an Antarctic Janthinobacterium sp. isolate:Evidence of a protease horizontal gene transfer event

    Cecilia Martinez-Rosales; Juan Jos Marizcurrena; Andrs Iriarte; Natalia Fullana; Hctor Musto; Susana Castro-Sowinski

    2015-01-01

    We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AU11 (growth between 4°C and 30°C) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98%query coverage) to subtilisin peptidases belonging to the S8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from S8A bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterium. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

  7. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases

    Batten, MR; Senior, BW; Kilian, Mogens;

    2003-01-01

    effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement......The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either...... side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial...

  8. Modulation of the Bacillus anthracis secretome by the immune inhibitor A1 protease.

    Pflughoeft, Kathryn J; Swick, Michelle C; Engler, David A; Yeo, Hye-Jeong; Koehler, Theresa M

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself. PMID:24214942

  9. Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases.

    Senior, Bernard W; Woof, Jenny M

    2005-03-01

    Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases. PMID:15731049

  10. Sites in the CH3 domain of human IgA1 that influence sensitivity to bacterial IgA1 proteases.

    Senior, Bernard W; Woof, Jenny M

    2006-09-15

    The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain. PMID:16951354

  11. Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.

    Lomholt, H; Poulsen, K; Kilian, M

    1995-02-01

    Cloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal alpha-peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N-terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic-like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies. PMID:7783620

  12. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

    Senior, BW; Batten, MR; Kilian, Mogens;

    2002-01-01

    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were...... constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases....

  13. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases.

    Senior, B W; Batten, M R; Kilian, M; Woof, J M

    2002-08-01

    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases. PMID:12196126

  14. Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

    Loechel, F; Overgaard, M T; Oxvig, C;

    1999-01-01

    prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition of the...... protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity of the...

  15. Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

    Aleshin,A.; Shiryaev, S.; Strongin, A.; Liddington, R.

    2007-01-01

    Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.

  16. Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases.

    Senior, B W; Dunlop, J I; Batten, M R; Kilian, M; Woof, J M

    2000-02-01

    To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition. PMID:10639405

  17. Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae

    De Paolis, Francesca; Beghetto, Elisa; Spadoni, Andrea; Montagnani, Francesca; Felici, Franco; Oggioni, Marco R; Gargano, Nicola

    2007-01-01

    Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery. PMID:18088426

  18. Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae

    Felici Franco

    2007-12-01

    Full Text Available Abstract Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.

  19. The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases.

    Senior, Bernard W; Woof, Jenny M

    2005-06-15

    The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc. PMID:15944283

  20. Studies on the gonococcal IgA1 protease II. Improved methods of enzyme purification and production of monoclonal antibodies to the enzyme.

    Blake, M S; Eastby, C

    1991-11-22

    Two types of extremely active proteases that cleave human IgA1 are produced by pathogenic Neisseria in minute concentrations. To study the antigenicity of these enzymes, a simplified method is described to purify these enzymes from large batch cultures to obtain a sufficient quantity of these IgA1 proteases to study these characteristics. In addition, we describe the production of both rabbit polyclonal and mouse monoclonal antibodies to one of these enzymes. One such monoclonal antibody seemed directed toward the active site of the IgA1 protease and inhibited its enzymatic activity. PMID:1960418

  1. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody.

    Ciferri, Claudio; Lipari, Michael T; Liang, Wei-Ching; Estevez, Alberto; Hang, Julie; Stawicki, Scott; Wu, Yan; Moran, Paul; Elliott, Mike; Eigenbrot, Charles; Katschke, Kenneth J; van Lookeren Campagne, Menno; Kirchhofer, Daniel

    2015-12-01

    High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition. PMID:26385991

  2. Immunoglobulins in nasal secretions of healthy humans: structural integrity of secretory immunoglobulin A1 (IgA1) and occurrence of neutralizing antibodies to IgA1 proteases of nasal bacteria

    Kirkeby, L; Rasmussen, TT; Reinholdt, Jesper;

    2000-01-01

    . Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11......). IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 x 10(3) to 7.2 x 10(6) CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, alpha...

  3. Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

    Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content of H. glycines female cysts (HglCE), and by the plant polyphenol epigallocatechin gallate (EGCG). General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EG...

  4. Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

    Pflughoeft, Kathryn J.; Swick, Michelle C.; Engler, David A.; Yeo, Hye-Jeong; Koehler, Theresa M.

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophores...

  5. Degradation of insulin by isolated mouse pancreatic acini. Evidence for cell surface protease activity

    In the present study, we have used isolated mouse pancreatic acini were used to investigate the relationship between 125I-insulin binding and its degradation in order to probe the nature and cellular localization of the degradative process. In these cells, the proteolysis of 125I-insulin was dependent on time and cell concentration, and was saturated by unlabeled insulin with a Km of 290 nM. Since this value was much higher than the Kd for insulin binding to its receptor (1.1 nM), the data indicated that 125I-insulin degradation by acini occurred primarily via nonreceptor mechanisms. Several lines of evidence suggested that insulin was being degraded by the neutral thiol protease, insulin degrading enzyme (IDE). First, insulin degradation was inhibited by thiolreacting agents such as N-ethylmaleimide and p-chloromercuribenzoate. Second, the Km for degradation in acini was similar to the reported Km for IDE in other tissues. Third, the enzyme activity had a relative mol wt of approximately 130,000 by gel filtration, a value similar to that reported for purified IDE. Fourth, the degrading activity was removed with a specific antibody to IDE. Other lines of evidence suggested that enzymes located on the cell surface played a role in insulin degradation by acini. First, the nonpenetrating sulfhydryl reacting agent 5,5' dithiobis-2-nitrobenzoic acid blocked 125I-insulin degradation. Second, a specific antibody to IDE identified the presence of the enzyme on the cell surface. Third, chloroquine, leupeptin and antipain, agents that inhibit lysosomal function, did not influence 125I-insulin degradation. Fourth, highly purified pancreatic plasma membranes degraded 125I-insulin

  6. The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR

    Kristian Tveten; Trine Ranheim; Knut Erik Berge; Trond P.Leren; Mari Ann Kulseth

    2009-01-01

    The low-density lipoprotein receptor (LDLR) mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins (LDLs). Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia (FH) is an autoso-mal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafilomycin Al or addition of the irreversible serine protease inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and 3,4-dichloroisocoumarin (DCI), prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1-positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.

  7. Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases

    Senior, B; Dunlop, JI; Batten, MR;

    2000-01-01

    melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required...

  8. Protease inhibitor

    2009-01-01

    The present invention relates to a polypeptide exhibiting a protease inhibitory activity and uses of said polypeptide in methods for inhibiting, directly or indirectly, one or more proteases of the blood clotting cascade. The invention also relates to use of said polypeptide as a pharmaceutical e...

  9. Processing Proteases

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused to the...... protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N-terminal of...... the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is...

  10. Supermarket Proteases.

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  11. Earthworm Protease

    The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  12. Structure-Function of Falcipains: Malarial Cysteine Proteases

    Pandey, Kailash C.; Rajnikant Dixit

    2012-01-01

    Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. ...

  13. No evidence for selection of HIV-1 with enhanced gag-protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.

    Denis R Chopera

    Full Text Available BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. METHODS AND RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.

  14. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    Rumlová, Michaela; Ruml, T.; Pohl, J.; Pichová, Iva

    2003-01-01

    Roč. 310, - (2003), s. 310-318. ISSN 0042-6822 R&D Projects: GA ČR GA203/00/1241; GA AV ČR IAB4055202 Institutional research plan: CEZ:AV0Z4055905 Keywords : M-PMV protease * HIV-1 capsid protein * HIV-1 protease Subject RIV: CE - Biochemistry Impact factor: 3.391, year: 2003

  15. Proteases as Insecticidal Agents

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic...

  16. Evidence for charged B meson decays to a1(1260)+/- pi0 and a1(1260)0 pi+/-

    Aubert, B; Boutigny, D; Karyotakis, Yu; Lees, J P; Poireau, V; Prudent, X; Tisserand, V; Zghiche, A; Garra Tico, J; Graugès-Pous, E; López, L; Palano, A; Eigen, G; Stugu, B; Sun, L; Abrams, G S; Battaglia, M; Brown, D N; Button-Shafer, J; Cahn, R N; Groysman, Y; Jacobsen, R G; Kadyk, J A; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; Lopes-Pegna, D; Lynch, G; Mir, L M; Orimoto, T J; Ronan, M T; Tackmann, K; Wenzel, W A; Del Amo-Sánchez, P; Hawkes, C M; Watson, A T; Held, T; Koch, H; Lewandowski, B; Pelizaeus, M; Schröder, T; Steinke, M; Walker, D; Asgeirsson, D J; Çuhadar-Dönszelmann, T; Fulsom, B G; Hearty, C; Mattison, T S; McKenna, J A; Khan, A; Saleem, M; Teodorescu, L; Blinov, V E; Bukin, A D; Druzhinin, V P; Golubev, V B; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Todyshev, K Yu; Bondioli, M; Curry, S; Eschrich, I; Kirkby, D; Lankford, A J; Lund, P; Mandelkern, M; Martin, E C; Stoker, D P; Abachi, S; Buchanan, C; Foulkes, S D; Gary, J W; Liu, F; Long, O; Shen, B C; Zhang, L; Paar, H P; Rahatlou, S; Sharma, V; Berryhill, J W; Campagnari, C; Cunha, A; Dahmes, B; Hong, T M; Kovalskyi, D; Richman, J D; Beck, T W; Eisner, A M; Flacco, C J; Heusch, C A; Kroseberg, J; Lockman, W S; Schalk, T; Schumm, B A; Seiden, A; Wilson, M G; Winstrom, L O; Chen, E; Cheng, C H; Fang, F; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Andreassen, R; Mancinelli, G; Meadows, B T; Mishra, K; Sokoloff, M D; Blanc, F; Bloom, P C; Chen, S; Ford, W T; Hirschauer, J F; Kreisel, A; Nagel, M; Nauenberg, U; Olivas, A; Smith, J G; Ulmer, K A; Wagner, S R; Zhang, J; Gabareen, A M; Soffer, A; Toki, W H; Wilson, R J; Winklmeier, F; Altenburg, D D; Feltresi, E; Hauke, A; Jasper, H; Merkel, J; Petzold, A; Spaan, B; Wacker, K; Klose, V; Kobel, M J; Lacker, H M; Mader, W F; Nogowski, R; Schubert, J; Schubert, K R; Schwierz, R; Sundermann, J E; Volk, A; Bernard, D; Bonneaud, G R; Latour, E; Lombardo, V; Thiebaux, C; Verderi, M; Clark, P J; Gradl, W; Muheim, F; Playfer, S; Robertson, A I; Xie, Y; Andreotti, M; Bettoni, D; Bozzi, C; Calabrese, R; Cecchi, A; Cibinetto, G; Franchini, P; Luppi, E; Negrini, M; Petrella, A; Piemontese, L; Prencipe, E; Santoro, V; Anulli, F; Baldini-Ferroli, R; Calcaterra, A; De Sangro, R; Finocchiaro, G; Pacetti, S; Patteri, P; Peruzzi, I M; Piccolo, M; Rama, M; Zallo, A; Buzzo, A; Contri, R; Lo Vetere, M; Macri, M M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Chaisanguanthum, K S; Morii, M; Wu, J; Dubitzky, R S; Marks, J; Schenk, S; Uwer, U; Bard, D J; Dauncey, P D; Flack, R L; Nash, J A; Panduro-Vazquez, W; Tibbetts, M; Behera, P K; Chai, X; Charles, M J; Mallik, U; Ziegler, V; Cochran, J; Crawley, H B; Dong, L; Eyges, V; Meyer, W T; Prell, S; Rosenberg, E I; Rubin, A E; Gao, Y Y; Gritsan, A V; Guo, Z J; Lae, C K; Denig, A G; Fritsch, M; Schott, G; Arnaud, N; Bequilleux, J; Davier, M; Grosdidier, G; Höcker, A; Lepeltier, V; Le Diberder, F; Lutz, A M; Pruvot, S; Rodier, S; Roudeau, P; Schune, M H; Serrano, J; Sordini, V; Stocchi, A; Wang, W F; Wormser, G; Lange, D J; Wright, D M; Bingham, I; Chavez, C A; Forster, I J; Fry, J R; Gabathuler, E; Gamet, R; Hutchcroft, D E; Payne, D J; Schofield, K C; Touramanis, C; Bevan, A J; George, K A; Di Lodovico, F; Menges, W; Sacco, R; Cowan, G; Flächer, H U; Hopkins, D A; Paramesvaran, S; Salvatore, F; Wren, A C; Brown, D N; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Chia, Y M; Edgar, C L; Lafferty, G D; West, T J; Yi, J I; Anderson, J; Chen, C; Jawahery, A; Roberts, D A; Simi, G; Tuggle, J M; Blaylock, G; Dallapiccola, C; Hertzbach, S S; Li, X; Moore, T B; Salvati, E; Saremi, S; Cowan, R; Dujmic, D; Fisher, P H; Koeneke, K; Sciolla, G; Sekula, S J; Spitznagel, M; Taylor, F; Yamamoto, R K; Zhao, M; Zheng, Y; Mclachlin, S E; Patel, P M; Robertson, S H; Lazzaro, A; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Côté, D; Simard, M; Taras, P; Viaud, F B; Nicholson, H; De Nardo, Gallieno; Fabozzi, F; Lista, L; Monorchio, D; Sciacca, C; Baak, M A; Raven, G; Snoek, H L; Jessop, C P; LoSecco, J M; Benelli, G; Corwin, L A; Honscheid, K; Kagan, H; Kass, R; Morris, J P; Rahimi, A M; Regensburger, J J; Wong, Q K; Blount, N L; Brau, J E; Frey, R; Igonkina, O; Kolb, J A; Lu, M; Rahmat, R; Sinev, N B; Strom, D; Strube, J; Torrence, E; Gagliardi, N; Gaz, A; Margoni, M; Morandin, M; Pompili, A; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Voci, C; Ben-Haim, E; Briand, H; Calderini, G; Chauveau, J; David, P; Del Buono, L; De La Vaissière, C; Hamon, O; Leruste, P; Malcles, J; Ocariz, J; Pérez, A; Gladney, L; Biasini, M; Covarelli, R; Manoni, E; Angelini, C; Batignani, G; Bettarini, S; Carpinelli, M; Cenci, R; Cervelli, A; Forti, F; Giorgi, M A; Lusiani, A; Marchiori, G; Mazur, M A; Morganti, M; Neri, N; Paoloni, E; Rizzo, G; Walsh, J J; Haire, M; Biesiada, J; Elmer, P; Lau, Y P; Lü, C; Olsen, J; Smith, A J S; Telnov, A V; Baracchini, E; Bellini, F; Cavoto, G; D'Orazio, A; Del Re, D; Di Marco, E; Faccini, R; Ferrarotto, F; Ferroni, F; Gaspero, M; Jackson, P D; Li Gioi, L; Mazzoni, M A; Morganti, S; Piredda, G; Polci, F; Renga, F; Voena, C; Ebert, M; Hartmann, T; Schröder, H; Waldi, R; Adye, T; Castelli, G; Franek, B; Olaiya, E O; Ricciardi, S; Röthel, W; Wilson, F F; Aleksan, R; Emery, S; Escalier, M; Gaidot, A; Ganzhur, S F; Hamel de Monchenault, G; Kozanecki, W; Vasseur, G; Yéche, C; Zito, M; Chen, X R; Liu, H; Park, W; Purohit, M V; Wilson, J R; Allen, M T; Aston, D; Bartoldus, R; Bechtle, P; Berger, N; Claus, R; Coleman, J P; Convery, M R; Dingfelder, J C; Dorfan, J; Dubois-Felsmann, G P; Dunwoodie, W; Field, R C; Glanzman, T; Gowdy, S J; Graham, M T; Grenier, P; Hast, C; Hrynóva, T; Innes, W R; Kaminski, J; Kelsey, M H; Kim, H; Kim, P; Kocian, M L; Leith, D W G S; Li, S; Luitz, S; Lüth, V; Lynch, H L; MacFarlane, D B; Marsiske, H; Messner, R; Müller, D R; O'Grady, C P; Ofte, I; Perazzo, A; Perl, M; Pulliam, T; Ratcliff, B N; Roodman, A; Salnikov, A A; Schindler, R H; Schwiening, J; Snyder, A; Stelzer, J; Su, D; Sullivan, M K; Suzuki, K; Swain, S K; Thompson, J M; Vavra, J; Van Bakel, N; Wagner, A P; Weaver, M; Wisniewski, W J; Wittgen, M; Wright, D H; Yarritu, A K; Yi, K; Young, C C; Burchat, P R; Edwards, A J; Majewski, S A; Petersen, B A; Wilden, L; Ahmed, S; Alam, M S; Bula, R; Ernst, J A; Jain, V; Pan, B; Saeed, M A; Wappler, F R; Zain, S B; Bugg, W; Krishnamurthy, M; Spanier, S M; Eckmann, R; Ritchie, J L; Ruland, A M; Schilling, C J; Schwitters, R F; Izen, J M; Lou, X C; Ye, S; Bianchi, F; Gallo, F; Gamba, D; Pelliccioni, M; Bomben, M; Bosisio, L; Cartaro, C; Cossutti, F; Della Ricca, G; Lanceri, L; Vitale, L; Azzolini, V; Lopez-March, N; Martínez-Vidal, F; Milanes, D A; Oyanguren, A; Albert, J; Banerjee, Sw; Bhuyan, B; Hamano, K; Kowalewski, R; Nugent, I M; Roney, J M; Sobie, R J; Harrison, P F; Ilic, J; Latham, T E; Mohanty, G B; Pappagallo, M; Band, H R; Chen, X; Dasu, S; Flood, K T; Hollar, J J; Kutter, P E; Pan, Y; Pierini, M; Prepost, R; Wu, S L; Neal, H

    2007-01-01

    We present measurements of the branching fractions for the decays B+/- --> a1(1260)+/- pi0 and B+/- --> a1(1260)0 pi+/- from a data sample of 232 * 10^6 BB pairs produced in e+e- annihilation through the Y(4S) resonance. We measure the branching fraction B(B+/- --> a1(1260)+/- pi0) * B(a1(1260)+/- --> pi- pi+ pi+/-) = (13.2 +/- 2.7 +/- 2.1) * 10^-6 with a significance of 4.2 sigma, and the branching fraction B(B+/- --> a1(1260)0 pi+/-) * B(a1(1260)0 --> pi- pi+ pi0) = (20.4 +/- 4.7 +/- 3.4) * 10^-6 with a significance of 3.8 sigma, where the first error quoted is statistical and the second is systematic.

  17. Evidence for the presence of phospholipase A1 in Saccharomyces cerevisiae

    The cause of the autolysis of pressed Baker's yeast was examined. Softened pressed yeast cells (Saccharomyces cerevisiae), after about 10 days of storage at 30 deg C, was subjected to a series of extraction: the extraction with acetone was made to the supernatant after the centrifugation of the water-suspended yeast cell at 1000 x g for 10 min, and the obtained precipitation was mechanically (with a Potter teflon homogenizer) homogenized. After removing the residues by centrifugation, the protein was salted out with ammonium sulfate up to 0.6 saturation. An enzyme, phospholipase A1 was thus obtained from the softened yeast cells. The activity of the enzyme thus obtained was assayed using L-α-phosphatidylethanolamine as the substrate. It was previously found that 14C-labelled free fatty acids liberated from phosphatidylcholine (PC) accumulated in the softened yeast packed cake. The enzyme was identified as phospholipase A1 having the optimal pH at around 8. Another evidence, obtained previously, together with the present finding suggest that the softening of the pressed Baker's yeast may be caused by the degradation of phospholipid by the combined action of phospholipase A1 and lysophospholipase L2. (Yamashita, S.)

  18. Proteases of neutrophilic granulocytes

    Wiesława Roszkowska-Jakimiec

    2002-06-01

    Full Text Available The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B, serine proteases (granulocytic elastase, cathepsin G, protease 3, membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine, cysteine and aspartic cathepsins. The role of these proteases in the pathology and diagnostics of certain diseases was considered.

  19. Proteases of neutrophilic granulocytes

    Wiesława Roszkowska-Jakimiec; Anna Worowska; Marek Gacko; Tomasz Maksimowicz

    2002-01-01

    The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B), serine proteases (granulocytic elastase, cathepsin G, protease 3), membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine), cysteine ...

  20. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

  1. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  2. Structure and function of ubiquitin: evidence for differential interactions of arginine-74 with the activating enzyme and the proteases of ATP-dependent proteolysis

    Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatography and subjected to tryptic peptide mapping to determine the location of the modification(s). These derivatives were stable throughout the procedures required for purification, tryptic hydrolysis, and peptide mapping. Derivative A was modified at arginine-42, derivative B at arginine-72, derivative C at arginines-42 and -72, and derivative D at arginine-74. Modification of ubiquitin with 14C-labeled 4-(oxoacetyl)phenoxyacetic acid indicated that the reagent formed a stable, 1:1 complex with arginine residues of the protein. Native ubiquitin and each of the four derivatives were tested for their ability to stimulate 32P exchange between ATP and pyrophosphate, a reaction catalyzed by enzyme 1 of the ubiquitin-dependent proteolytic pathway. A and C were capable of promoting this exchange at a rate only 15% that of native ubiquitin, B stimulated the exchange to 25%, and D stimulated exchange to 60% of the native level. None of the derivative was capable of promoting a significant level of ubiquitin-dependent proteolysis. These results indicate that in this system, the integrity or arginines-42, -72, and -74 is essential for full function of ubiquitin and suggest that the ubiquitin activating enzyme (E1) and the protease(s) of the system recognize different regions or conformations of ubiquitin

  3. Commercial proteases: present and future.

    Li, Qing; Yi, Li; Marek, Peter; Iverson, Brent L

    2013-04-17

    This review presents a brief overview of the general categories of commercially used proteases, and critically surveys the successful strategies currently being used to improve the properties of proteases for various commercial purposes. We describe the broad application of proteases in laundry detergents, food processing, and the leather industry. The review also introduces the expanding development of proteases as a class of therapeutic agents, as well as highlighting recent progress in the field of protease engineering. The potential commercial applications of proteases are rapidly growing as recent technological advances are producing proteases with novel properties and substrate specificities. PMID:23318711

  4. Evidence of association with type 1 diabetes in the SLC11A1 gene region

    Walker Neil M; Stevens Helen E; Nutland Sarah; Howson Joanna MM; Downes Kate; Yang Jennie HM; Todd John A

    2011-01-01

    Abstract Background Linkage and congenic strain analyses using the nonobese diabetic (NOD) mouse as a model for human type 1 autoimmune diabetes (T1D) have identified several NOD mouse Idd (insulin dependent diabetes) loci, including Slc11a1 (formerly known as Nramp1). Genetic variants in the orthologous region encompassing SLC11A1 in human chromosome 2q35 have been reported to be associated with various immune-related diseases including T1D. Here, we have conducted association analysis of th...

  5. Effect of Legionella pneumophila cytotoxic protease on human neutrophil and monocyte function

    Rechnitzer, C; Kharazmi, A

    1992-01-01

    protease on the chemotactic activity of neutrophils was demonstrated by the continued inhibition of neutrophil chemotaxis when the protease was removed following pre-incubation of the cells. In contrast, the enzyme had no effect on monocyte chemotaxis. The protease inhibited, also in a concentration...... activity of L. pneumophila protease on neutrophil chemotaxis and on the listericidal activity of human neutrophils and monocytes demonstrated in this study provides evidence for a role of this enzyme in the pathogenesis of Legionnaires' disease....

  6. Evidence for quark-hadron duality in the proton spin asymmetry A1

    Spin-dependent lepton-nucleon scattering data have been used to investigate the validity of the concept of quark-hadron duality for the spin asymmetry A1. Longitudinally polarised positrons were scattered off a longitudinally polarised hydrogen target for values of Q2 between 1.2 and 12 GeV2 and values of W2 between 1 and 4 GeV2. The average double-spin asymmetry in the nucleon resonance region is found to agree with that measured in deep-inelastic scattering at the same values of the Bjorken scaling variable x. This finding implies that the description of A1 in terms of quark degrees of freedom is valid also in the nucleon resonance region for values of Q2 above 1.6 GeV2. (orig.)

  7. Structure-Function of Falcipains: Malarial Cysteine Proteases

    Kailash C. Pandey

    2012-01-01

    Full Text Available Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. falciparum. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. Overall, the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria. Drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies.

  8. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

    Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of...

  9. Bacterial proteases and virulence

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  10. Nucleic Acid Aptamers Against Proteases

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø; Andreasen, P A

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of...

  11. Enteroviral proteases: structure, host interactions and pathogenicity.

    Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Nurminen, Anssi; Hytönen, Vesa P; Flodström-Tullberg, Malin

    2016-07-01

    Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145174

  12. Death proteases come alive

    Woltering, E.J.

    2004-01-01

    Cell death in plants exhibits morphological features comparable to caspase-mediated apoptosis in animals, suggesting that plant cell death is executed by (caspase-like) proteases. However, to date, no caspase homologues have been identified in plants and therefore the existence and nature of these p

  13. Proteases in Periodontal Disease

    Ana Rita Sokolonski ANTON

    2006-09-01

    Full Text Available Introduction: The caries and the periodontal disease (PD are the most frequent alterations in the oral cavity. The PD presents two stages: gengivitis and periodontitis. The destruction of collagenous fibers which encases the tooth onto the alveolar bone is characteristic of the pariodontitis. The inclusion loss caused by this pathology is due to the presence of bacteria and their products, besides the tissue destruction. This process is caused by excessive discharge of cells of the organism defence which reach the damaged area, and among these cells are neutrophils. These cells free lysosomal granule, where enzymes known as proteases (elastase, colagenasis and catepsin G are present. When excessively delivered, they cause extensive tissue destruction. The organism innate defence respond to this process activating anti-proteases, such as alfa-1-antitripsin e alfa-2-macrogoblulin, and, as consequence, the inflammatory process is subdued. Objective: Revision of the literature on periodontitis and its markers. In periodontitis, the balance between protease and anti-protese seems to be altered and lead to the appearance of these ones. There is an increase of prevalence of PD in the world population. In recent times, it has been associated to systemic conditions that lead to tissue destruction. Perhaps, the cause is based on an exacerbated tissue reaction, more than on the bacterial aggression. Conclusion: The predisposition of the organism is an important factor for the disease development. At reading different studies, it was observed that the discharged protease during the neutrophils degranulation process has internal, not bacterial, origin.

  14. Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.

    Duggal, Ruchia; Liu, Yilin; Gregory, Michael C; Denisov, Ilia G; Kincaid, James R; Sligar, Stephen G

    2016-08-19

    Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to

  15. Induction of CYP26A1 by Metabolites of Retinoic Acid: Evidence That CYP26A1 Is an Important Enzyme in the Elimination of Active Retinoids

    Topletz, Ariel R.; Tripathy, Sasmita; Foti, Robert S.; Shimshoni, Jakob A.; Nelson, Wendel L.

    2015-01-01

    All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARβ were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD+, suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 μl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action. PMID:25492813

  16. Cathepsin proteases in Toxoplasma gondii

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and n...

  17. Nucleic Acid Aptamers Against Proteases

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø;

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  18. Substrate modulation of enzyme activity in the herpesvirus protease family

    Lazic, Ana; Goetz, David H.; Nomura, Anson M.; Marnett, Alan B.; Craik, Charles S.

    2007-01-01

    The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 Å resolution structure of Kaposi’s Sarcoma herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 Å2 of surface are buried in the newly formed S4 pocket when tyrosin...

  19. Protease-mediated drug delivery

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  20. Can UGT1A1 genotyping reduce morbidity and mortality in patients with metastatic colorectal cancer treated with irinotecan? An evidence-based review

    Palomaki, Glenn E; Bradley, Linda A.; Douglas, Michael P.; Kolor, Katherine; Dotson, W. David

    2009-01-01

    This evidence-based review addresses the question of whether testing for UGT1A1 mutations in patients with metastatic colorectal cancer treated with irinotecan leads to improvement in outcomes (e.g., irinotecan toxicity, response to treatment, morbidity, and mortality), when compared with no testing. No studies were identified that addressed this question directly. The quality of evidence on the analytic validity of current UGT1A1 genetic testing methods is adequate (scale: convincing, adequa...

  1. Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and Bacillus cereus producing protease bacillolysins and exo-chitinases

    Tomas eErban

    2016-02-01

    Full Text Available Tyrophagus putrescentiae (Schrank, 1781 is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida. In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities and mite-bacterial interaction in dry dog food. Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30 and (polyubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (dry dog food and low-fat, low-protein (flour diets to 1% and 5% (w/w, and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist

  2. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases.

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  3. Evident?

    Plant, Peter

    2012-01-01

    Quality assurance and evidence in career guidance in Europe are often seen as self-evident approaches, but particular interests lie behind......Quality assurance and evidence in career guidance in Europe are often seen as self-evident approaches, but particular interests lie behind...

  4. Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

    Alkaline protease is one of the most important enzymes in industry, medicine, and research. In the present work, a comparative detection for alkaline protease activity was established for instant detection of enzyme activity. Eight different alkalophilic bacterial isolates were compared based on the clear zone they produced on skim milk agar. One strain gave an absolute clear zone in 16 hours and was used for alkaline protease detection. The result of Phenotypic identification using Biology Microlog 3 identified the isolate as Exiguobacterium acetylicum. The isolate under study showed slightly different characteristics from a known Exiguobacterium acetylicum strain. The isolate tolerated alkaline conditions up to ph 11, while good growth was evident at ph 7, the maximum alkaline protease activity was observed at ph 9 which reached up to 109.01 U/ml. The alkaline activity assay using alkaline protease enzyme assay were coordinating with those obtained by conductivity; there was a relevant decrease in conductivity at the maximum increase in enzyme activity, which proved the cell membrane conductivity has a close relation to alkaline protease production. This isolate has tolerated gamma radiation, the increase in dose (up to 4 Gy) gave wider clear zones in terms of diameter and this was relevant to the conductivity measurements

  5. Protease-Sensitive Synthetic Prions

    Colby, David W; Rachel Wain; Baskakov, Ilia V.; Giuseppe Legname; Palmer, Christina G.; Nguyen, Hoang-Oanh B.; Azucena Lemus; Cohen, Fred E.; Stephen J DeArmond; Prusiner, Stanley B.

    2010-01-01

    Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, ...

  6. Evidence against the structural gene encoding type II collagen (COL2A1) as the mutant locus in achondroplasia.

    Ogilvie, D.; Wordsworth, P; Thompson, E.; Sykes, B

    1986-01-01

    The structure of the locus encoding the major cartilage collagen gene (COL2A1) was studied in a total of 19 cases of achondroplasia. No gross rearrangements were seen. The segregation of COL2A1 was examined in three affected kindreds using restriction site and length variants as genetic markers. In two kindreds discordant segregation between the achondroplasia and COL2A1 loci was demonstrated. Paternity/maternity was confirmed using a 'minisatellite' core sequence probe which reveals cross hy...

  7. Serine proteases of parasitic helminths.

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  8. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic ana...

  9. Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and Bacillus cereus producing protease bacillolysins and exo-chitinases

    Tomas eErban; Dagmar eRybanska; Karel eHarant; Bronislava eHortova; Jan eHubert

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities and mite-bacterial interaction in dry dog food. Proteomic methods comprising enzymatic and zymographic analysis o...

  10. Serine Proteases of Parasitic Helminths

    Yong YANG; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we...

  11. Does Protease-Antiprotease Imbalance Explain Chronic Obstructive Pulmonary Disease?

    Lomas, David A

    2016-04-01

    Chronic obstructive pulmonary disease (COPD) is defined as airflow limitation that is not fully reversible. The airflow limitation is usually progressive and is associated with the inhalation of noxious gases, typically cigarette smoke. The protease-antiprotease paradigm suggests that the pathogenesis of COPD and emphysema is the result of an imbalance between enzymes that degrade the extracellular matrix within the lung and proteins that oppose this proteolytic activity. This review assesses the genetic evidence in support of protease-antiprotease imbalance in the pathogenesis of COPD. It also articulates why suppression of protease activity in alpha-1 antitrypsin deficiency may be insufficient to prevent the progression of COPD. Rather, alpha-1 antitrypsin deficiency may be better treated by small-molecules so reads molecules, RNA-silencing, and other strategies that target the protein misfolding and polymerization that cause the disease. PMID:27115947

  12. Cysteine proteases: mode of action and role in epidermal differentiation.

    Brocklehurst, Keith; Philpott, Mike P

    2013-02-01

    Desquamation or cell shedding in mammalian skin is known to involve serine proteases, aspartic proteases and glycosidases. In addition, evidence continues to accumulate that papain-like cysteine proteases and an inhibitor cystatin M/E largely confined to the cutaneous epithelia also play key roles in the process. This involves the complete proteolysis of cell adhesive structures of the stratum corneum, the corneodesmosomes and notably of the desmogleins. Continual cell replacement in the epidermis is the result of the balance between the loss of the outer squames and mitosis of the cells in the basal cell layer. This article provides a brief account of the salient features of the characteristics and catalytic mechanism of cysteine proteases, followed by a discussion of the relevant epidermal biology. The proteases include the asparaginyl endopeptidase legumain, which exerts a strict specificity for the hydrolysis of asparaginyl bonds, cathepsin-V and cathepsin-L. The control of these enzymes by cystatin M/E regulates the processing of transglutaminases and is crucial in the biochemical pathway responsible for regulating the cross-linking and desquamation of the stratum corneum. In addition, caspase-14 has now been shown to play a major part in epidermal maturation. Uncontrolled proteolytic activity leads to abnormal hair follicle formation and deleterious effects on the skin barrier function. PMID:23344364

  13. Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

    Lukomski, S; Sreevatsan, S; Amberg, C; Reichardt, W.; Woischnik, M.; Podbielski, A; Musser, J M

    1997-01-01

    Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mut...

  14. Microbial inhibitors of cysteine proteases.

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  15. Unraveling the roles of Atg4 proteases from autophagy modulation to targeted cancer therapy.

    Zhang, Lan; Li, Jingjing; Ouyang, Liang; Liu, Bo; Cheng, Yan

    2016-04-01

    Atg4 proteases are cysteine proteases, which are well known for their crucial roles in the lipidation and delipidation of LC3 during the autophagy process. At least four human Atg4 homologs have been reported according to their sequence homology to the yeast Saccharomyces cerevisiae (Sc) Atg4. Accumulating evidence has recently indicated that abnormal expression levels of some human Atg4 proteins occur in several types of cancer cells, which may be closely related to tumor progression, tumor suppression and cancer therapy resistance. In this review, we focus on highlighting the pivotal roles of the human Atg4 proteases in the LC3 conjugation system and their major function in autophagy. Moreover, we further explore the roles of human Atg4 proteases as potential novel targets for cancer therapy. Taken together, these findings would shed light on elucidating the key roles of Atg4 proteases from autophagy modulation to targeted cancer therapy. PMID:26805760

  16. Cytomegalovirus protease targeted prodrug development.

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable. PMID:23485093

  17. Enzymatic Degradation of Ovalbumin by Various Proteases

    Matsumoto, Kiyoshi; Yoshimaru, Tetsuro; Matsui, Toshiro; Osajima, Yutaka

    1997-01-01

    An investigation was made of the enzymatic hydrolysis of ovalbumin (OVA), a major allergen in egg white, by various acid and alkaline proteases. Protease YP-SS (acid protease) from Aspergillus niger and alcalase (alkaline protease) from BacilLus licheniformis were found to be useful for the degradation of OVA, respectively. OVA was almost totally hydrolyzed within 15 hr at 37℃ by alcalase. Alcalase acted rapidly to hydrolyze OVA, with about 90% of OVA being hydrolyzed within 30min., the react...

  18. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  19. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity

  20. Curcumin derivatives as HIV-1 protease inhibitors

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  1. Exogenous proteases for meat tenderization.

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  2. Comparative Studies on Retroviral Proteases: Substrate Specificity

    József Tözsér

    2010-01-01

    Full Text Available Exogenous retroviruses are subclassified into seven genera and include viruses that cause diseases in humans. The viral Gag and Gag-Pro-Pol polyproteins are processed by the retroviral protease in the last stage of replication and inhibitors of the HIV-1 protease are widely used in AIDS therapy. Resistant mutations occur in response to the drug therapy introducing residues that are frequently found in the equivalent position of other retroviral proteases. Therefore, besides helping to understand the general and specific features of these enzymes, comparative studies of retroviral proteases may help to understand the mutational capacity of the HIV-1 protease.

  3. ATP-dependent protease in maize mitochondria

    ATP-dependent protease was identified in the matrix of Zea mays L. Sachara mitochondria. 14C-methylated casein has been used as a substrate, and the matrix ATP-dependent protease exhibited similar sensitivity towards specific inhibitors as the Lon protease from E. coli nd analogues from rat liver and yeast mitochondria. Here we report the existence of Lon like ATP-dependent protease in intact mitochondria prepared from 4-days-old epicotyls of Zea mays L. seedling. Enzyme has been purified from Lubrol treated mitochondria using ion exchange chromatography and gel filtration. The enzyme activity has been estimated using 14C-methylated casein as a substrate and sensitivity of the protease towards the specific inhibitors has been tested. ATP-dependent protease from the mitochondrial matrix of maize exhibit similar sensitivity to the above mentioned inhibitors like Lon protease from yeast and rat liver mitochondria as well as from E. coli. (authors)

  4. Biotechnology of Cold-Active Proteases

    Tulasi Satyanarayana

    2013-05-01

    Full Text Available The bulk of Earth’s biosphere is cold (<5 °C and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  5. Production of secretory leucocyte protease inhibitor (SLPI) in human pancreatic beta-cells.

    Nyström, M; Bergenfeldt, M; Ljungcrantz, I.; Lindeheim, A; Ohlsson, K.

    1999-01-01

    Secretory leucocyte protease inhibitor (SLPI) is a potent inhibitor of granulocyte elastase and cathepsin G, and also an inhibitor of pancreatic enzymes like trypsin, chymotrypsin and pancreatic elastase. SLPI has also been shown to inhibit HIV-1 infections by blocking viral DNA synthesis. Since SLPI is an inhibitor of pancreatic proteases we wished to investigate whether SLPI was also actually produced in the pancreas. M-RNA from human pancreatic tissue showed evidence of SLPI production usi...

  6. Production of Secretory Leucocyte Protease Inhibitor (SLPI) in Human Pancreatic β-Cells

    Max Nyström; Magnus Bergenfeldt; Irena Ljungcrantz; Èsa Lindeheim; Kjell Ohlsson

    1999-01-01

    Secretory leucocyte protease inhibitor (SLPI) is a potent inhibitor of granulocyte elastase and cathepsin G, and also an inhibitor of pancreatic enzymes like trypsin, chymotrypsin and pancreatic elastase. SLPI has also been shown to inhibit HIV-1 infections by blocking viral DNA synthesis. Since SLPI is an inhibitor of pancreatic proteases we wished to investigate whether SLPI was also actually produced in the pancreas. M-RNA from human pancreatic tissue showed evidence of SLPI production usi...

  7. Plant caspase-like proteases in plant programmed cell death

    Xu, Qixian; Zhang, Lingrui

    2009-01-01

    Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, ...

  8. A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

    Takayuki Shindo

    Full Text Available Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa. In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

  9. Protease gene families in Populus and Arabidopsis

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  10. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.; Chorna V. I.

    2011-01-01

    The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic anal...

  11. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  12. Protease-sensitive synthetic prions.

    David W Colby

    2010-01-01

    Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

  13. A Phytophthora infestans Cystatin-Like Protein Targets a Novel Tomato Papain-Like Apoplastic Protease1[W][OA

    Tian, Miaoying; Win, Joe; Song, Jing; van der Hoorn, Renier; van der Knaap, Esther; Kamoun, Sophien

    2007-01-01

    There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions. PMID:17085509

  14. Antimicrobial proteins and peptides in human lung diseases: A friend and foe partnership with host proteases.

    Lecaille, Fabien; Lalmanach, Gilles; Andrault, Pierre-Marie

    2016-03-01

    Lung antimicrobial proteins and peptides (AMPs) are major sentinels of innate immunity by preventing microbial colonization and infection. Nevertheless bactericidal activity of AMPs against Gram-positive and Gram-negative bacteria is compromised in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and asthma. Evidence is accumulating that expression of harmful human serine proteases, matrix metalloproteases and cysteine cathepsins is markedely increased in these chronic lung diseases. The local imbalance between proteases and protease inhibitors compromises lung tissue integrity and function, by not only degrading extracellular matrix components, but also non-matrix proteins. Despite the fact that AMPs are somewhat resistant to proteolytic degradation, some human proteases cleave them efficiently and impair their antimicrobial potency. By contrast, certain AMPs may be effective as antiproteases. Host proteases participate in concert with bacterial proteases in the degradation of key innate immunity peptides/proteins and thus may play immunomodulatory activities during chronic lung diseases. In this context, the present review highlights the current knowledge and recent discoveries on the ability of host enzymes to interact with AMPs, providing a better understanding of the role of human proteases in innate host defense. PMID:26341472

  15. Advances in protease engineering for laundry detergents.

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. PMID:25579194

  16. Extracellular proteases as targets for drug development.

    Cudic, Mare; Fields, Gregg B

    2009-08-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV) and cysteine proteases (cathepsin B) are discussed herein. PMID:19689354

  17. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H.; Roush, William R.

    2008-01-01

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely r...

  18. A biotechnology perspective of fungal proteases

    Paula Monteiro Souza; Mona Lisa de Assis Bittencourt; Carolina Canielles Caprara; Marcela de Freitas; Renata Paula Coppini de Almeida; Dâmaris Silveira; Yris Maria Fonseca; Edivaldo Ximenes Ferreira Filho; Adalberto Pessoa Junior; Pérola Oliveira Magalhães

    2015-01-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy...

  19. Extracellular proteases as targets for drug development

    Cudic, Mare; Fields, Gregg B.

    2009-01-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addit...

  20. Thioamide-Based Fluorescent Protease Sensors

    Goldberg, Jacob M.; Chen, Xing; Meinhardt, Nataline; Greenbaum, Doron C.; Petersson, E. James

    2014-01-01

    Thioamide quenchers can be paired with compact fluorophores to design “turn-on” fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually an...

  1. Biased Signaling of Protease-activated Receptors

    PeishenZhao; NigelWilliamBunnett

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an e...

  2. Protein targeting to ATP-dependent proteases

    Inobe, Tomonao; Matouschek, Andreas

    2008-01-01

    ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be u...

  3. Proteolytic crosstalk in multi-protease networks

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  4. Evidence for defect-induced superconductivity up to 49 K in (C a1 -xRx) F e2A s2

    Deng, L. Z.; Lv, B.; Zhao, K.; Wei, F. Y.; Xue, Y. Y.; Wu, Z.; Chu, C. W.

    2016-02-01

    To explore the origin of the unusual nonbulk superconductivity with a Tc up to 49 K reported in the rare-earth-doped CaF e2A s2 , the chemical composition, magnetization, specific heat, resistivity, and annealing effect are systematically investigated on nominal (C a1 -xRx) F e2A s2 single crystals with different x and R =La , Ce, Pr, and Nd. All display a doping-independent Tc once superconductivity is induced, a doping-dependent low field superconducting volume fraction f , and a large magnetic anisotropy η in the superconducting state, suggesting a rather inhomogeneous superconducting state in an otherwise microscale homogenous superconductor. The wavelength dispersive spectroscopy and specific heat show the presence of defects that are closely related to f , regardless of the R involved. The magnetism further reveals that the defects are mainly superparamagnetic clusters for R =Ce , Pr, and Nd with strong intercluster interactions, implying that defects are locally self-organized. Annealing at 500 °C, without varying the doping level x , suppresses f profoundly but not the Tc. The above observations provide evidence for the crucial role of defects in the occurrence of the unusually high Tc˜49 K in (C a1 -xRx) F e2A s2 and are consistent with the interface-enhanced superconductivity recently proposed.

  5. Extracellular and membrane-bound proteases from Bacillus subtilis.

    Mäntsälä, P; Zalkin, H

    1980-01-01

    Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The e...

  6. Immobilization to prevent enzyme incompatibility with proteases

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was s

  7. Production of alkaline protease from Cellulosimicrobium cellulans

    Luciana Ferracini-Santos; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p

  8. Progress and prospects on DENV protease inhibitors.

    Timiri, Ajay Kumar; Sinha, Barij Nayan; Jayaprakash, Venkatesan

    2016-07-19

    New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B-NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective. PMID:27092412

  9. HIV-1 protease mutations and protease inhibitor cross-resistance.

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W

    2010-10-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  10. Compensatory substitutions in the HCV NS3/4A protease cleavage sites are not observed in patients treated unsuccessfully with telaprevir combination treatment

    Sullivan James C

    2012-08-01

    Full Text Available Abstract Background Development of compensatory mutations within the HIV p7/p1 and p1/p6 protease cleavage site region has been observed in HIV-infected patients treated with protease inhibitors. Mechanisms of fitness compensation may occur in HCV populations upon treatment of HCV protease inhibitors as well. Findings In this study, we investigated whether substitutions in protease cleavage site regions of HCV occur in response to a treatment regimen containing the NS3/4A protease inhibitor telaprevir (TVR. Evaluation of viral populations from 569 patients prior to treatment showed that the four NS3/4A cleavage sites were well conserved. Few changes in the cleavage site regions were observed in the 159 patients who failed TVR combination treatment, and no residues displayed evidence of directional selection after the acquisition of TVR-resistance. Conclusions Cleavage site mutations did not occur after treatment with the HCV protease inhibitor telaprevir.

  11. Proteases at work: Cues for understanding neural development and degeneration

    Paul Saftig

    2015-05-01

    Full Text Available Proteolytical processing of membrane bound molecules is a fundamental mechanism for the degradation of these proteins as well as for controlling cell-to-cell communication, which is at the basis of tissue development and homeostasis. Members of families of metalloproteinases and intra-membrane proteases are major effectors of these events. A recent workshop in Baeza, Spain, was devoted to discuss how this mechanism coordinates brain development and how its dysfunction leads to brain pathologies. Herein we summarize the findings presented during this workshop, which illuminate the role of metalloproteinases, including MMPs, ADAM-proteases and intra-membrane proteases, in the regulation of neurogenesis, axon guidance and synaptogenesis as well as in neurodegeneration. Indeed, there is increasing evidence that proteolysis at the membrane is directly linked to neuropathologies such as Alzheimer Disease and autism spectrum or prion disorders. These proteolytic events are tightly regulated and we are just at the beginning of understanding how these processes could be exploited to design therapeutic treatments aimed at alleviating psychiatric and neurodegenerative pathologies.

  12. Mast cell proteases as pharmacological targets.

    Caughey, George H

    2016-05-01

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  13. Caspase Family Proteases and Apoptosis

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  14. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    Werner Smidt

    Full Text Available The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1 infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS. Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  15. Natural inhibitors of tumor-associated proteases

    The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

  16. Fatores antinutricionais: inibidores de proteases e lectinas Antinutritional factors: protease inhibitors and lectins

    Mara Reis SILVA

    2000-04-01

    Full Text Available Os fatores antinutricionais presentes em alimentos podem provocar efeitos fisiológicos adversos ou diminuir a biodisponibilidade de nutrientes. A maior questão sobre os riscos à saúde provocados por antinutrientes é o desconhecimento dos níveis de tolerância, do grau de variação do risco individual e da influência de fatores ambientais sobre a capacidade de detoxificação do organismo humano. Dentre os fatores antinutricionais os inibidores de proteases e as lectinas são considerados instáveis ao tratamento térmico. A hipertrofia pancreática causada pelos inibidores de tripsina tem sido relatada em alguns estudos com animais. As alterações da função fisiológica em animais causadas por ação de lectinas no intestino parecem estar relacionadas à especificidade destas substâncias com as células da mucosa intestinal. Os possíveis efeitos adversos dos inibidores de proteases e das lectinas na maioria das vezes são inferidos somente de experimentos com animais de laboratório.The antinutritional factors present in foods can cause adverse physiological effects or decrease the bioavailability of nutrients. Health risk factors associated with antinutrients include: lack of knowledge of the tolerance levels to these compounds in the human organism, little available information on the degree of variation of individual risks and little knowledge with respect to the influence of environmental factors on the detoxification capacity of the human organism. The possible adverse effects of protease inhibitors and lectin on human health are, in most cases, only inferred by way of experiments with laboratory animals. Pancreatic hypertrophy caused by trypsin inhibitors has been shown in some animal experiments. Alterations in physiological functions at the intestinal level shown by animals submitted to lectins containing diets seem to be related to the specificity of these substances with the intestinal mucosa cells. There is no evidence

  17. A Serine Protease Homolog Negatively Regulates TEP1 Consumption in Systemic Infections of the Malaria Vector Anopheles gambiae

    Yassine, Hassan; Kamareddine, Layla; Chamat, Soulaima; Christophides, George K.; Osta, Mike A.

    2014-01-01

    Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of Anopheles gambiae, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negat...

  18. Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

    Madhusudhan Budatha; Simone Silva; Teodoro Ignacio Montoya; Ayako Suzuki; Sheena Shah-Simpson; Cecilia Karin Wieslander; Masashi Yanagisawa; Ruth Ann Word; Hiromi Yanagisawa

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic tryp...

  19. Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities

    Mielech, Anna M.; Chen, Yafang; MESECAR, Andrew D.; Baker, Susan C.

    2014-01-01

    Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome cor...

  20. Lung protease/anti-protease network and modulation of mucus production and surfactant activity.

    Garcia-Verdugo, Ignacio; Descamps, Delphyne; Chignard, Michel; Touqui, Lhousseine; Sallenave, Jean-Michel

    2010-11-01

    Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local 'alarm' anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs. PMID:20493919

  1. Antimalarial Synergy of Cysteine and Aspartic Protease Inhibitors

    Semenov, Andrey; Olson, Jed E.; Rosenthal, Philip J.

    1998-01-01

    It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease ...

  2. Synthesis of amino heterocycle aspartyl protease inhibitors.

    Chambers, Rachel K; Khan, Tanweer A; Olsen, David B; Sleebs, Brad E

    2016-06-14

    Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds. PMID:27143279

  3. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    Sojka, Daniel; FRANCISCHETTI, IVO M. B.; Calvo, Eric; KOTSYFAKIS, MICHALIS

    2011-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model...

  4. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R; Simmons, Graham

    2015-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coron...

  5. Electrically sensing protease activity with nanopores

    Kukwikila, Mikiembo; Howorka, Stefan

    2010-11-01

    The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

  6. Extracellular acid proteases produced by Saccharomycopsis lipolytica.

    T. Yamada; Ogrydziak, D M

    1983-01-01

    Saccharomycopsis lipolytica CX161-1B produced at least three extracellular acid proteases during exponential growth in medium containing glycerol, Difco Proteose Peptone, and mineral salts at pH 3.4 (Difco Laboratories, Detroit, Mich.). Little extracellular acid protease activity was produced with glutamic acid as the sole nitrogen source, somewhat higher levels were obtained with peptone, and much higher levels were obtained with Difco Proteose Peptone. The relative amounts of the three prot...

  7. A radiometric assay for HIV-1 protease

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources

  8. ADAM Proteases and Gastrointestinal Function.

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  9. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  10. Protease inhibitors targeting coronavirus and filovirus entry.

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H; Renslo, Adam R; Simmons, Graham

    2015-04-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  11. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes [3H]casein to acid-soluble products in the presence of ATP (or dATP) and Mg2+. Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti

  12. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon- cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

  13. Evolution of the paralogous hap and iga genes in Haemophilus influenzae: evidence for a conserved hap pseudogene associated with microcolony formation in the recently diverged Haemophilus aegyptius and H. influenzae biogroup aegyptius

    Kilian, Mogens; Poulsen, Knud; Lomholt, Hans Bredsted

    2002-01-01

    the mechanisms of evolution of two paralogous genes, hap and iga, which encode the adhesion and penetration Hap protein and the IgA1 protease respectively. Partial sequencing of hap and iga genes in a comprehensive collection of strains belonging to the H. influenzae/H. aegyptius complex revealed...... as revealed by phylogenetic analysis. There was no evidence for a second, functional copy of the hap gene in these strains. The perturbed expression of the Hap serine protease appears to be associated with the formation of elongated bacterial cells growing in chains and a distinct colonization...

  14. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-01

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties. PMID:27387593

  15. Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

    Although protease E was isolated from human pancreas over 10 years ago, its amino acid sequence and relationship to the elastases have not been established. The authors report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. They have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. They also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1

  16. Identification of covalent active site inhibitors of dengue virus protease

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  17. Identification of covalent active site inhibitors of dengue virus protease.

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  18. BACTERIAL ISOLATES OF MARINE COAST AS COMMERCIAL PRODUCER OF PROTEASE

    S. Das

    2012-01-01

    Full Text Available The objective of this work was to explore and exploit the extracellular protease secreting marine microbial biodiversity of the eastern coastal region of India. Culture dependent method was applied for isolation of microbes from the marine coast of West Bengal (Digha and Mandarmani and Andhra Pradesh (Vizag in India. Six protease secreting isolates were screened using casein hydrolysing property as well as azocasein assay and characterized on the basis of their morphological, biochemical, physiological and 16S rDNA based molecular properties. The enzymes were used for various commercial applications at a laboratory scale. Besides milk media and Luria Bertini broth, all the isolates grew in carbon minimal salt medium with jaggeri or tamarind as the carbon source (0.3% w/v. They showed intracellular metal accumulation when grown in presence of metal salts in the medium as evident from Energy Dispersive X-ray Fluorescense Analysis (EDXRF data. Maximum accumulation of lead was found in case of Bacillus cereus SM2. It showed equal efficiency of metal removal from solid strip at zero valent state. The isolates were also capable of complete removal of silver from exposed X-ray film after 48 hrs of incubation except for Escherichia coli SD1. Bacillus cereus SM2, isolate SD2 (closest to Bacillus pumilus and isolate SV1 (closest to Bacillus cibi were able to enhance the cleaning efficiency of detergent when used as additive. Use of tamarind and jaggeri as carbon source in minimal medium would make the process cost effective during large scale application. The ability to grow in a wide range of temperature and pH and accumulation of heavy metals revealed that these isolates would be potential candidates for bioremediation. Thus the marine diversity for protease production is extremely rich with immense commercial applications.

  19. Subtilisin-like proteases in nematodes.

    Poole, Catherine B; Jin, Jingmin; McReynolds, Larry A

    2007-09-01

    Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes. PMID:17570539

  20. Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes

    Lorenzi, Adriana Sturion; Silva, Genivaldo Gueiros Z.; Lopes, Fabyano Alvares Cardoso; Chia, Mathias Ahii; Edwards, Robert A.

    2016-01-01

    Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes). PMID:27151783

  1. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

    En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner.......En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner....

  2. Activation of ADAM 12 protease by copper

    Loechel, F; Wewer, Ulla M.

    2001-01-01

    Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...

  3. Hydrolysis of Fish Protein by Analkaline Protease

    2001-01-01

    Cod muscle protein was hydrolyzed by an alkaline protease in our study. The influences of hydrolysis temperature,fish protein concentration,and ratio of protease addition to protein amount on its degree of hy drolysis (DH) of protein were studied in details by applying dual quadratic rotary combinational design. The final results showed that more than 84% cod muscle protein could be hydrolyzed and recovered. Cod protein hydrolysate thus obtained had a balanced amino acid composition and mainly consisted of small peptides with molecule weight less than 6900 dalton.

  4. Detection of protease and protease activity using a single nanoscrescent SERS probe

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  5. Importance of lysosomal cysteine proteases in lung disease

    Chapman Harold A; Wolters Paul J

    2000-01-01

    Abstract The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically ...

  6. Centenary celebrations article: Cysteine proteases of human malaria parasites

    Pandey, Kailash C.

    2011-01-01

    There is an urgent need for new drugs against malaria, which takes millions of lives annually. Cysteine proteases are potential new drug targets, especially when current drugs are showing resistance. Falcipains and vivapains are well characterized cysteine proteases of P. falciparum and P. vivax, respectively. Studies with cysteine protease inhibitors and manipulating cysteine proteases specific genes have suggested their roles in hemoglobin hydrolysis. In P. falciparum, falcipain-2 and falci...

  7. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

    Wigdal, Susan S.; Anderson, Jessica L; Vidugiris, Gediminas J; Shultz, John; Wood, Keith V.; Fan, Frank

    2008-01-01

    Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily...

  8. High Throughput Substrate Phage Display for Protease Profiling

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W.

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imagin...

  9. The Role of Protease Activity in ErbB Biology

    Blobel, Carl P; Carpenter, Graham; Freeman, Matthew

    2008-01-01

    Proteases are now recognized as having an active role in a variety of processes aside from their recognized metabolic role in protein degradation. Within the ErbB system of ligands and receptors proteases are known to be necessary for the generation of soluble ligands from transmembrane precursers and for the processing of the ErbB4 receptor, such that its intracellular domain is translocated to the nucleus. There are two protease activities involved in the events: proteases that cleave withi...

  10. Comparative Study of Dermatophytic Fungi for Extra Cellular Proteases Efficacy

    Sanchita Chaturvedi; Sonal Pathak; Ruchi Upadhyay; Shweta Dubey

    2013-01-01

    Fungi are known to produce proteases of different kind. The dermatphytic fungal strains were isolated from human skin tissues for extra cellular proteases efficacy. The present study deals with purification, estimation and comparison of extracellular proteases from five fungal species. (Fusarium sp., Curvularia sp. , Fumigatus Sp. , Aspergillus Sp. and Mucor Sp.). All the five fungal strains showed good amount of extra cellular protease activity in terms of unit total protein content. By test...

  11. Predicting protein function from structure: Unique structural features of proteases

    Stawiski, Eric W.; Baucom, Albion E.; Lohr, Scott C.; Gregoret, Lydia M.

    2000-01-01

    We have noted consistent structural similarities among unrelated proteases. In comparison with other proteins of similar size, proteases have smaller than average surface areas, smaller radii of gyration, and higher Cα densities. These findings imply that proteases are, as a group, more tightly packed than other proteins. There are also notable differences in secondary structure content between these two groups of proteins: proteases have fewer helices and more loops. We speculate that both h...

  12. Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

    Abian, Olga; Vega, Sonia; Sancho, Javier; Velazquez-Campoy, Adrian

    2013-01-01

    The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease. PMID:23936097

  13. Overexpression of Elastin Affects the Protease to Anti-Protease Balance and Protects Mice from Colitis. : Elafin protects from colitis

    Motta*, Jean-Paul; Magne, Laurent; Descamps, Delphyne; Rolland, Corinne; Squarzoni-Dale, Camila; Rousset, Perrine; Balloy, Viviane; Huerre, Michel; Jenne, Dieter; Wartelle, Julien; Belaaouaj, Azzaq; Mas, Emmanuel; Vinel, Jean-Pierre; Alric, Laurent; Chignard, Michel

    2010-01-01

    BACKGROUND & AIMS:: Colon tissues of patients with inflammatory bowel disease (IBD) have been reported to have increased proteolytic activity, but no studies have clearly addressed the protease to anti-protease balance in the pathogenesis of colitis. We investigated the role of Elafin, a serine protease inhibitor expressed by skin and mucosal surfaces in human inflammatory conditions, and the proteases neutrophil elastase (NE) and proteinase-3 (PR-3), in mice with colitis. METHODS:: We studie...

  14. MICROSPHERE-BASED FLOW CYTOMETRY PROTEASE ASSAYS FOR USE IN PROTEASE ACTIVITY DETECTION AND HIGH-THROUGHPUT SCREENING

    Saunders, Matthew J.; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

    2010-01-01

    This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein specific protease of interest and results can be measured in both real time and as end point fluorescence assays on a flow cytometer. End point assays...

  15. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

    Kousted, Tina M; Skjødt, Karsten; Petersen, Steen V;

    2014-01-01

    , conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all...... serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition...... abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the...

  16. cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): Evidence for rapid evolutionary divergence of the gene

    Li, Kehua; Christiano, A.M.; Chu, Mon Li; Uitto, J. (Jefferson Medical College, Philadelphia, PA (United States) Thomas Jefferson Univ., Philadelphia, PA (United States)); Copeland, N.G.; Gilbert, D.J. (NCI-Federick Cancer Research and Development Center, Federick, MD (United States))

    1993-06-01

    Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study the authors have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of [approximately]9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5[prime] half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7Al. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. 33 refs., 5 figs., 1 tab.

  17. HIV-1 protease-induced apoptosis

    Rumlová, Michaela; Křížová, Ivana; Keprová, Alena; Hadravová, Romana; Doležal, Michal; Strohalmová, Karolína; Pichová, Iva; Hájek, Miroslav; Ruml, T.

    2014-01-01

    Roč. 11, May 20 (2014), 37/1-37/15. ISSN 1742-4690 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : HIV protease * BCA3 * AKIP-1 * apoptosis * mitochondria Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/37

  18. Proteases decode the extracellular matrix cryptome.

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-03-01

    The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets. PMID:26382969

  19. Intracellular aspartic protease of Candida albicans

    Bauerová, Václava; Pichová, Iva; Hrušková-Heidingsfeldová, Olga

    Mátraháza : -, 2007. s. 43. [Alexander Von Humboldt Workshop on Structure Based Approaches Towards Disease Control. 22.05.2007-27.05.2007, Mátraháza] Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * intracellular * aspartic protease Subject RIV: CE - Biochemistry

  20. A new class of SUMO proteases

    Gillies, Jennifer; Hochstrasser, Mark

    2012-01-01

    A new class of SUMO protease, DeSUMOylating enzyme (DeSI)—that has different substrates and localization to SENP SUMO proteases—is characterized in this issue of EMBO reports. The implications for the field are discussed here.

  1. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    Luca Musante; Dorota Tataruch; Dongfeng Gu; Xinyu Liu; Carol Forsblom; Per-Henrik Groop; Harry Holthofer

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profi...

  2. A novel protease activity assay using a protease-responsive chaperone protein

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  3. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs: Biogenesis and Function

    Nathalie Dautin

    2010-05-01

    Full Text Available Serine Protease Autotransporters of Enterobacteriaceae (SPATEs constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.

  4. New directions for protease inhibitors directed drug discovery.

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  5. Kinetic Solvent Isotope Effect in Human P450 CYP17A1 Mediated Androgen Formation: Evidence for a Reactive Peroxoanion Intermediate

    Gregory, Michael C.; Denisov, Ilia G.; Grinkova, Yelena V.; Khatri, Yogan; Sligar, Stephen G.

    2013-01-01

    Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the Cytochrome P450 superfamily. In the formation of the male hormone dehydroepiandrosterone, pregnenolone is first hydroxylated by P450 CYP17A1 at the 17-carbon followed a second round of catalysis by the same enzyme that cleaves C17–C20 carbon-carbon bond releasing acetic acid and the 17-keto product. In order to explore the mechani...

  6. NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex*

    Kim, Young Mee; Gayen, Shovanlal; Kang, CongBao; Joy, Joma; Huang, Qiwei; Chen, Angela Shuyi; Wee, John Liang Kuan; Ang, Melgious Jin Yan; Lim, Huichang Annie; Hung, Alvin W.; Li, Rong; Noble, Christian G.; Lee, Le Tian; Yip, Andy; Wang, Qing-Yin; Chia, Cheng San Brian; Hill, Jeffrey; Shi, Pei-Yong; Keller, Thomas H.

    2013-01-01

    The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker (“linked protease”), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a 1H-15N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV. PMID:23511634

  7. Identification of Genotypic Changes in Human Immunodeficiency Virus Protease That Correlate with Reduced Susceptibility to the Protease Inhibitor Lopinavir among Viral Isolates from Protease Inhibitor-Experienced Patients

    Kempf, Dale J.; Isaacson, Jeffrey D.; King, Martin S.; Brun, Scott C.; Xu, Yi; Real, Kathryn; Bernstein, Barry M.; Japour, Anthony J.; Sun, Eugene; Rode, Richard A

    2001-01-01

    The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with ...

  8. Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

    Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I

    2016-05-01

    The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. PMID:27041592

  9. Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

    Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. These results demonstrate that all the insulin cleavage sites generated by the Drosopohila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function

  10. Mechanism of oxidative inactivation of human presequence protease (hPreP) by hydrogen peroxide

    Chen, Jue; Teixeira, Pedro Filipe; Glaser, Elzbieta; Levine, Rodney L.

    2014-01-01

    The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β-peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to...

  11. Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation.

    Tiaden, André N; Bahrenberg, Gregor; Mirsaidi, Ali; Glanz, Stephan; Blüher, Matthias; Richards, Peter J

    2016-06-01

    Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions. Stem Cells 2016;34:1601-1614. PMID:26864869

  12. Novel peptide-based protease inhibitors

    Roodbeen, Renée

    This thesis describes the design and synthesis of peptide-based serine protease inhibitors. The targeted protease, urokinase-type plasminogen activator (uPA) activates plasminogen, which plays a major role in cancer metastasis. The peptide upain-2 (S 1 ,S 12-cyclo-AcCSWRGLENHAAC-NH2) is a highly...... specific inhibitor of uPA. With the aim of creating better inhibitors based on the upain-2 scaffold, the following three strategies were explored: First, it was attempted to predefine the structure of upain-2 in solution by incorporating turn-inducing sequences and peptidomimetics. Additionally...... bond across the ring. The second bridge was made by a disulfide bridge, amide bond formation or via ring-closing metathesis. A, with upain-2 equipotent, bicyclic inhibitor was obtained and its binding to uPA was studied by ITC, NMR and X-ray. The knowledge of how selective inhibitors bind uPA has been...

  13. Lipase and protease extraction from activated sludge

    Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.;

    2003-01-01

    gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination for......In the process of wastewater treatment hydrolysis of polymeric substances is the first and rate-limiting step. A closer study of the enzymes catalysing these reactions is essential for a better understanding of the microbial activity in the wastewater treatment process. Therefore, development of...... the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase...

  14. Protease gene shuffling and expression in Pichia pastoris

    Gang Yang

    2015-06-01

    Full Text Available Four kinds of neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis were isolated and shuffled. The shuffled genes were selected, inserted into pGAPZαA plasmid and transformed into Escherichia coli. The gene which could express high-activity protease was selected by screening the sizes of transparent zones around the colonies on casein plates. After an ideal protease gene was selected, it was sequenced and then transformed into Pichia pastoris X33. The result showed that the base in 1022th position of shuffled protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the mutant or native protease genes, the mutant protease activity was 36.4% higher than the native protease (P<0.05. The optimal pH and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease (P<0.05.

  15. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  16. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

  17. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    Madhusudhan Budatha

    Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

  18. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Mi Kyung Park

    Full Text Available Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25 in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  19. Protease Activation and Inflammation in Acute Pancreatitis

    Regnér, Sara

    2008-01-01

    Approximately 10—20 % of patients with acute pancreatitis (AP) develop a severe disease with high mortality and morbidity. Activation of pancreatic proteases, the inflammatory response and impaired pancreatic circulation are pathophysiological events that are important in order for the disease to develop. There is no specific treatment for severe AP, and no useful marker for predicting the severity of the disease upon admission to the hospital. In this thesis, markers of early pathophysio...

  20. Targeting exosites on blood coagulation proteases

    Monteiro, Robson Q.

    2005-01-01

    The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed ...

  1. Luminometric method for screening retroviral protease inhibitors

    Horáková, D.; Rumlová, Michaela; Pichová, Iva; Ruml, Tomáš

    2005-01-01

    Roč. 345, č. 1 (2005), s. 96-101. ISSN 0003-2697 R&D Projects: GA AV ČR(CZ) IAA4055304; GA MŠk(CZ) 1M0508; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506 Keywords : retroviral protease * inhibitors * luminescent assay Subject RIV: CE - Biochemistry Impact factor: 2.670, year: 2005

  2. Corruption of Innate Immunity by Bacterial Proteases

    Potempa, Jan; Pike, Robert N.

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host’s innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections. PMID:19756242

  3. Mitochondrial Proteases as Emerging Pharmacological Targets.

    Gibellini, Lara; De Biasi, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello

    2016-01-01

    The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions. PMID:26831646

  4. Clinical utility of serine proteases in breast cancer

    The serine protease uPA and its inhibitor PAI-1 are involved in the degradation of tumor stroma and basement membrane. The independent prognostic value of serine protease urokinase-type plasminogen activator uPA and its inhibitor PAI-1 in breast cancer has been almost uniformly confirmed in numerous individual studies as well as in a meta-analysis, including 18 data sets of more than 8,000 patients. According to these observations, the risk of relapse in node negative patients with low levels of uPA and PAI-1 is less then 10%; these patients could be spared from toxic adjuvant systemic therapy. Clinically relevant and even more important is the information that uPA and its inhibitor PAI-1 may also have a predictive value for response to either hormonal or cytotoxic therapy in early breast cancer. According to our data obtained from altogether 460 operable breast cancer patients, uPA and PAI-1 may have a predictive value for the response to hormone therapy, but not to chemotherapy. The high PAI-1 levels were associated with a higher risk of relapse in the patients without adjuvant systemic therapy (HR 2.14; C.I. 95%0.48-9.56; p=0.321) and in the patients treated with chemotherapy (RR 2.48; C.I. 95%= 1.35-4.57; p=0.003). However, in the patients treated with adjuvant hormone therapy, either alone or in combination with chemotherapy, the prognostic value of uPA and PAI-1 was diminished. Moreover, high levels of both uPA and PAI-1 were associated with a lower risk of relapse (HR 0.79; p=0.693 and HR 0.26 p= 0.204, respectively). On the basis of currently available evidence, serine protease uPA and its inhibitor PAI-1 are certainly the markers that improve a proper selection of candidates for adjuvant systemic therapy and may also be the markers that could facilitate treatment decision in each individual patient, which is of utmost importance. (author)

  5. High throughput substrate phage display for protease profiling.

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  6. Serum albumin as a probe for testing the selectivity of irreversible cysteine protease inhibitors: The case of vinyl sulfones.

    Regazzoni, Luca; Colombo, Simone; Mazzolari, Angelica; Vistoli, Giulio; Carini, Marina

    2016-05-30

    Vinyl sulfones are used for drug design of irreversible inhibitors of cysteine proteases since they are able to alkylate cysteine thiols inside the catalytic pocket of this class of enzymes. Some authors have reported the lack of reactivity towards glutathione as sufficient evidence of the selectivity of such a mechanism. Herein, we demonstrate that some simple molecules containing a vinyl sulfone moiety are not thiol-specific alkylants since they react with some albumin nucleophiles including side chains of Cys34 and His146. Such side-reactions are not desirable for any drug candidate since they limit serum stability, bioavailability and they possibly trigger toxicity mechanisms. In silico predictions, indicate that the compounds tested share similar structural features with reported inhibitors of cysteine proteases, as well as similar poses around the main albumin nucleophiles. Altogether, the data suggest that albumin is better than glutathione for the setup of early in vitro tests probing the selectivity of cysteine protease inhibitors. PMID:26970985

  7. Structural basis for HTLV-1 protease inhibition by the HIV-1 protease inhibitor indinavir.

    Kuhnert, Maren; Steuber, Holger; Diederich, Wibke E

    2014-07-24

    HTLV-1 protease (HTLV-1 PR) is an aspartic protease which represents a promising drug target for the discovery of novel anti-HTLV-1 drugs. The X-ray structure of HTLV-1 PR in complex with the well-known and approved HIV-1 PR inhibitor Indinavir was determined at 2.40 Å resolution. In this contribution, we describe the first crystal structure in complex with a nonpeptidic inhibitor that accounts for rationalizing the rather moderate affinity of Indinavir against HTLV-1 PR and provides the basis for further structure-guided optimization strategies. PMID:25006983

  8. Temporal dependence of cysteine protease activation following excitotoxic hippocampal injury

    Berry, Jennifer N.; Sharrett-Field, Lynda; Butler, Tracy R.; Prendergast, Mark A.

    2012-01-01

    Excitotoxic insults can lead to intracellular signaling cascades that contribute to cell death, in part by activation of proteases, phospholipases, and endonucleases. Cysteine proteases, such as calpains, are calcium-activated enzymes which degrade cytoskeletal proteins, including microtubule-associated proteins, tubulin, and spectrin, among others. The current study used the organotypic hippocampal slice culture model to examine whether pharmacologic inhibition of cysteine protease activity ...

  9. Cold-Adapted Proteases as an Emerging Class of Therapeutics

    Fornbacke, Marcus; Clarsund, Mats

    2013-01-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments—cold-adapted or psychrophilic proteases—generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible...

  10. Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-01-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individua...

  11. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

    Nilesh P. Nirmal; R. Seeta Laxman

    2014-01-01

    A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found ...

  12. Erwinia carotovora extracellular proteases : characterization and role in soft rot

    Kyöstiö, Sirkka R. M.

    1990-01-01

    Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene enc...

  13. Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.; Bolon, Daniel N. A.; Schiffer, Celia A.

    2012-01-01

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Di...

  14. Molecular dynamics simulations of HIV-1 protease complexed with saquinavir

    Watson, S. J.

    2009-01-01

    Inhibition of the Human Immunode�ficiency virus type-1 (HIV-1) protease enzyme blocks HIV-1 replication. Protease inhibitor drugs have successfully been used as a therapy for HIV-infected individuals to reduce their viral loads and slow the progression to Acquired Immune Defi�ciency Syndrome (AIDS). However, mutations readily and rapidly accrue in the protease gene resulting in a reduced sensitivity of the protein to the inhibitor. In this thesis, molecular dynamics simulations (MDS)...

  15. Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database

    Shafer, Robert W.; Jung, Duane R.; Betts, Bradley J.; Xi, Yinong; Gonzales, Matthew J.

    2000-01-01

    The HIV RT and Protease Sequence Database is an online relational database that catalogs evolutionary and drug-related human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease sequence variation (http://hivdb.stanford.edu ). The database contains a compilation of nearly all published HIV RT and protease sequences including International Collaboration database submissions (e.g., GenBank) and sequences published in journal articles. Sequences are linked to data about the sourc...

  16. Protein protease inhibitors in insects and comparison with mammalian inhibitors.

    Eguchi, M

    1993-01-01

    1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results. PMID:8365101

  17. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Suk Ho Choi

    2011-01-01

    Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inh...

  18. A preliminary neutron diffraction analysis of Achromobacter protease I

    Ohnishi, Yuki; Masaki, Takeharu; Yamada, Taro; Kurihara, Kazuo; Tanaka, Ichiro; Niimura, Nobuo

    2010-11-01

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  19. A preliminary neutron diffraction analysis of Achromobacter protease I

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  20. A functional proteomics screen of proteases in colorectal carcinoma.

    McKerrow, J H; Bhargava, V.; Hansell, E.; Huling, S.; Kuwahara, T.; Matley, M.; Coussens, L; Warren, R

    2000-01-01

    BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver me...

  1. Self and non-self discrimination by "restriction proteases".

    Lefkovits, I

    1986-01-01

    I propose that an organism possesses a set of specific enzymes ("restriction proteases") that cleave self proteins at defined amino acid sequences unless these sequences are rendered inaccessible by glycosylation. Intracellular proteins are degraded by restriction proteases when cells die. In this way, intracellular proteins remain undetected by the immune system. I propose that some autoimmune diseases are caused by the absence of a specific restriction protease.

  2. Isolation of a Tomato Protease that May Be Involved in Proteolysis of 1-Aminocyclopropane-1-Carboxylate Synthase

    Jian-Feng LI; Liang-Hu QU; Ning LI

    2005-01-01

    1-aminocyclopropane-1-carboxylate (ACC) synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase (LeACS2) in tomato (Lycopersicon esculentum Mill.) fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ℃. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthaseprocessing protease from plant tissues.

  3. HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses.

    Velasco-Arroyo, Blanca; Diaz-Mendoza, Mercedes; Gandullo, Jacinto; Gonzalez-Melendi, Pablo; Santamaria, M Estrella; Dominguez-Figueroa, Jose D; Hensel, Goetz; Martinez, Manuel; Kumlehn, Jochen; Diaz, Isabel

    2016-07-01

    Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth. PMID:27217548

  4. Role of Protease-Inhibitors in Ocular Diseases

    Nicola Pescosolido

    2014-12-01

    Full Text Available It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI, metalloproteinase inhibitor (TIMP, maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI, and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models.

  5. Induction of Protease Activity in Vibrio anguillarum by Gastrointestinal Mucus

    Denkin, Steven M.; Nelson, David R.

    1999-01-01

    The effect of gastrointestinal mucus on protease activity in Vibrio anguillarum was investigated. Protease activity was measured by using an azocasein hydrolysis assay. Cells grown to stationary phase in mucus (200 μg of mucus protein/ml) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Protease induction was examined with cells grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-...

  6. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Suk Ho Choi

    2011-09-01

    Full Text Available Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1 The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2 The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3 Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II chloride inhibited it. 4 The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions: The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

  7. Hordeum vulgare cysteine protease heterologous expressed in yeast

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach;

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the...... active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. One of the key cysteine proteases in Barley...

  8. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  9. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  10. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group

  11. Series of HIV-1 protease nanomolar inhibitors; binding to WT and mutant protease

    Skálová, Tereza; Dohnálek, Jan; Dušková, Jarmila; Petroková, Hana; Vondráčková, Eva; Hašek, Jindřich

    Florence : International Union of Crystallography, 2005. C243. [Congress of the International Union of Crystallography /20./. 23.8.2005-31.8.2005, Florence] R&D Projects: GA AV ČR(CZ) KJB4050312 Keywords : HIV retroviral proteases * HIV drug design * macromolecular crystal structure Subject RIV: EB - Genetics ; Molecular Biology

  12. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. PMID:27028647

  13. Production and cleavage specificity determination of serine proteases mMCP-4, mMCP-5, rMCP-2 and two platypus serine proteases of the chymase locus.

    Sidibeh, Cherno Omar

    2013-01-01

    Serine proteases are a family of enzymes with a wide array of functions across both eukaryotes and prokaryotes. Here we have attempted to produce the serine proteases rat mast cell protease 2 and mouse mast cell protease 5 in a culture of HEK 293 cells; and mouse mast cell protease 4, platypus granzyme B-like protease and platypus hypothetical protease in a baculovirus expression system. Following production we wanted to analyse these serine proteases using a phage display assay and a battery...

  14. A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L. syn Senna tora (L. Roxb. seed extract

    Garg Satyendra K

    2011-07-01

    Full Text Available Abstract Background Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. Methods The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90% followed by dialysis and size exclusion chromatography (SEC. The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60% and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD and ANOVA were employed as statistical tools. Results The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD of protease

  15. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain fam...

  16. A review on production of serine alkaline protease by Bacillus spp

    Biswanath Bhunia; Bikram Basak; Apurba Dey

    2012-01-01

    In recent times, protease has gained considerable importance in the world market. Proteases are groups of proteins included in the subclass hydrolases, within the main class enzymes. Serine alkaline proteases (SAP) are one of the most important groups of industrial enzymes. They account for approximately 35% of the total microbial enzyme sales. Serine protease is produced by various types of fermentation techniques using microorganism. Among the proteases, bacterial proteases are more signifi...

  17. Intramembrane Proteolysis by Signal Peptide Peptidases: A Comparative Discussion of GXGD-type Aspartyl Proteases*

    Fluhrer, Regina; Steiner, Harald; Haass, Christian

    2009-01-01

    Intramembrane-cleaving proteases are required for reverse signaling and membrane protein degradation. A major class of these proteases is represented by the GXGD-type aspartyl proteases. GXGD describes a novel signature sequence that distinguishes these proteases from conventional aspartyl proteases. Members of the family of the GXGD-type aspartyl proteases are the Alzheimer disease-related γ-secretase, the signal peptide peptidases and their homologs, and the bacteria...

  18. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta.

    Cao, Xiaolong; He, Yan; Hu, Yingxia; Zhang, Xiufeng; Wang, Yang; Zou, Zhen; Chen, Yunru; Blissard, Gary W; Kanost, Michael R; Jiang, Haobo

    2015-07-01

    Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species. PMID:25530503

  19. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  20. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B;

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and...

  1. Effect of proteases on the β-thromboglobulin radioimmunoassay

    Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of β-thromboglobulin and platelet factor 4. The initial assays indicated that a β-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the β-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of α1-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases. 24 references, 2 figures, 4 tables

  2. A NOVEL APPROACH TO REGULATE NITROGEN MINERALIZATION USING PROTEASE INHIBITORS

    Mineralization of organic N sources by extracellular proteases affects both the availability of inorganic N to plants and losses of N to the environment. We hypothesized that (i) application of purified protease inhibitors would slow down soil N mineralization, and (ii) elevated concentrations of pr...

  3. Isolation and characterization of proteases from Bacteroides melaninogenicus.

    Fujimura, S.; Nakamura, T.(International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan)

    1981-01-01

    We isolated two types of intracellular proteases from a strain of Bacteroides melaninogenicus. These enzymes were extracted from cells by ultrasonic treatment and were partially purified. These two enzymes (proteases I and II) differed in molecular weight, heat stability, sensitivity to reducing agents, Km value, and optimum pH for activity.

  4. Cathepsin Protease Inhibition Reduces Endometriosis Lesion Establishment.

    Porter, Kristi M; Wieser, Friedrich A; Wilder, Catera L; Sidell, Neil; Platt, Manu O

    2016-05-01

    Endometriosis is a gynecologic disease characterized by the ectopic presence of endometrial tissue on organs within the peritoneal cavity, causing debilitating abdominal pain and infertility. Current treatments alleviate moderate pain symptoms associated with the disorder but exhibit limited ability to prevent new or recurring lesion establishment and growth. Retrograde menstruation has been implicated for introducing endometrial tissue into the peritoneal cavity, but molecular mechanisms underlying attachment and invasion are not fully understood. We hypothesize that cysteine cathepsins, a group of powerful extracellular matrix proteases, facilitate endometrial tissue invasion and endometriosis lesion establishment in the peritoneal wall and inhibiting this activity would decrease endometriosis lesion implantation. To test this, we used an immunocompetent endometriosis mouse model and found that endometriotic lesions exhibited a greater than 5-fold increase in active cathepsins compared to tissue from peritoneal wall or eutopic endometrium, with cathepsins L and K specifically implicated. Human endometriosis lesions also exhibited greater cathepsin activity than adjacent peritoneum tissue, supporting the mouse results. Finally, we tested the hypothesis that inhibiting cathepsin activity could block endometriosis lesion attachment and implantation in vivo. Intraperitoneal injection of the broad cysteine cathepsin inhibitor, E-64, significantly reduced the number of attached endometriosis lesions in our murine model compared to vehicle-treated controls demonstrating that cathepsin proteases contribute to endometriosis lesion establishment, and their inhibition may provide a novel, nonhormonal therapy for endometriosis. PMID:26482207

  5. Purification and Characterization of An Alkaline Protease from Acetes chinensis

    XU Jiachao; LIU Xin; LI Zhaojie; XU Jie; XUE Changhu; GAO Xin

    2005-01-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55 ℃ and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+ , EDTA and PMSF could inhibit its activity.

  6. Alkaline Protease Production by a Strain of Marine Yeasts

    WANG Ping; CHI Zhenming; MA Chunling

    2006-01-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China.The protease had the highest activity at pH 9.0 and 45 ℃.The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0.The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min-1.Under the optimal conditions, 623.1 Umg-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

  7. Comparative Study of Dermatophytic Fungi for Extra Cellular Proteases Efficacy

    Sanchita Chaturvedi

    2013-07-01

    Full Text Available Fungi are known to produce proteases of different kind. The dermatphytic fungal strains were isolated from human skin tissues for extra cellular proteases efficacy. The present study deals with purification, estimation and comparison of extracellular proteases from five fungal species. (Fusarium sp., Curvularia sp. , Fumigatus Sp. , Aspergillus Sp. and Mucor Sp.. All the five fungal strains showed good amount of extra cellular protease activity in terms of unit total protein content. By testing all the crude extracts for enzyme activity, Fusarium sp. was found to show the highest activity whereas Mucor sp. showed the lowest. The study supports the notion that fungi can be a good source of extracellular enzymes especially proteases.

  8. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  9. The family of Deg/HtrA proteases in plants

    Schuhmann Holger

    2012-04-01

    Full Text Available Abstract Background The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required. Results Using phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a “core set” of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell. Conclusions In our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.

  10. Phase-Variable Expression of an Operon Encoding Extracellular Alkaline Protease, a Serine Protease Homolog, and Lipase in Pseudomonas brassicacearum

    Chabeaud, Philippe; de Groot, Arjan; Bitter, Wilbert; Tommassen, Jan; Heulin, Thierry; Achouak, Wafa

    2001-01-01

    The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

  11. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family.

    Tanz, Sandra K; Castleden, Ian; Hooper, Cornelia M; Small, Ian; Millar, A Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1-Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  12. Sweet potato cysteine proteases SPAE and SPCP2 participate in sporamin degradation during storage root sprouting.

    Chen, Hsien-Jung; Liang, Shu-Hao; Huang, Guan-Jhong; Lin, Yaw-Huei

    2015-08-15

    Sweet potato sporamins are trypsin inhibitors and exhibit strong resistance to digestion by pepsin, trypsin and chymotrypsin. In addition, they constitute the major storage proteins in the sweet potato and, after degradation, provide nitrogen as a nutrient for seedling regrowth in sprouting storage roots. In this report, four cysteine proteases-one asparaginyl endopeptidase (SPAE), two papain-like cysteine proteases (SPCP1 and SPCP2), and one granulin-containing cysteine protease (SPCP3)-were studied to determine their association with sporamin degradation in sprouting storage roots. Sporamin degradation became significant in the flesh of storage roots starting from week 4 after sprouting and this correlated with expression levels of SPAE and SPCP2, but not of SPCP1 and SPCP3. In the outer flesh near the skin, sporamin degradation was more evident and occurred earlier than in the inner flesh of storage roots. Degradation of sporamins in the outer flesh was inversely correlated with the distance of the storage root from the sprout. Exogenous application of SPAE and SPCP2, but not SPCP3, fusion proteins to crude extracts of the outer flesh (i.e., extracted from a depth of 0.3cm and within 2cm of one-week-old sprouts) promoted in vitro sporamin degradation in a dose-dependent manner. Pre-treatment of SPAE and SPCP2 fusion proteins at 95°C for 5min prior to their application to the crude extracts reduced sporamin degradation. These data show that sweet potato asparaginyl endopeptidase SPAE and papain-like cysteine protease SPCP2 participate in sporamin degradation during storage root sprouting. PMID:26363719

  13. Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process

    Catherine S. Adamson

    2012-01-01

    Full Text Available Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR, which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

  14. Reaction mechanism of -acylhydroxamate with cysteine proteases

    R Shankar; P Kolandaivel

    2007-09-01

    The gas-phase reaction mechanism of -acylhydroxamate with cysteine proteases has been investigated using ab initio and density functional theory. On the irreversible process, after breakdown of tetrahedral intermediate (INT1), small 1-2 anionotropic has been formed and rearranged to give stable by-products sulfenamide (P1) and thiocarbamate (P2) with considerable energy loss. While, on the reversible part of this reaction mechanism, intermediate (INT2) breaks down on oxidation, to form a stable product (P3). Topological and AIM analyses have been performed for hydrogen bonded complex in this reaction profile. Intrinsic reaction coordinates [IRC, minimum-energy path (MEP)] calculation connects the transition state between R-INT1, INT1-P1 and INT1-P2. The products P1, P2 and P3 are energetically more stable than the reactant and hence the reaction enthalpy is found to be exothermic.

  15. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  16. Cold-adapted proteases as an emerging class of therapeutics.

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications. PMID:25135820

  17. Identification of covalent active site inhibitors of dengue virus protease

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: Dengue virus (DENV protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  18. Fluorometric CCHFV OTU protease assay with potent inhibitors.

    Kocabas, Fatih; Aslan, Galip S

    2015-10-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay. PMID:26156848

  19. Exploring a new serine protease from Cucumis sativus L.

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345

  20. Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

    E. V. Маtseliukh

    2015-04-01

    Full Text Available Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. plantarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physico-chemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metal-dependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 °C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen. These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

  1. Laundry detergent compatibility of the alkaline protease from Bacillus cereus.

    Banik, Rathindra Mohan; Prakash, Monika

    2004-01-01

    The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C. PMID:15293947

  2. Structure of HIV-1 protease determined by neutron crystallography

    HIV-1 protease is an aspartic protease, and plays an essential role in replication of HIV. To develop HIV-1 protease inhibitors through structure-based drug design, it is necessary to understand the catalytic mechanism and inhibitor recognition of HIV-1 protease. We have determined the crystal structure of HIV-1 protease in complex with KNI-272 to 1.9 A resolution by neutron crystallography in combination with 1.4 A resolution X-ray diffraction data. The results show that the carbonyl group of hydroxymethylcarbonyl (HMC) in KNI-272 forms a hydrogen bonding interaction with protonated Asp 25 and the hydrogen atom from the hydroxyl group of HMC forms a hydrogen bonding interaction with the deprotonated Asp125. This is the first neutron report for HIV-1/inhibitor complex and shows directly the locations of key hydrogen atoms in catalysis and in the binding of a transition-state analog. The results confirm key aspect of the presumed catalytic mechanism of HIV-1 protease and will aid in the further development of protease inhibitors. (author)

  3. Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

    Little Tom J

    2009-06-01

    Full Text Available Abstract Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the An. gambiae species complex in both East and West Africa. Results Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes. Conclusion It is well known that phylogenetic and population history in the An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed.

  4. A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins.

    Pomerantsev, Andrei P; Pomerantseva, Olga M; Moayeri, Mahtab; Fattah, Rasem; Tallant, Cynthia; Leppla, Stephen H

    2011-11-01

    Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1⁺, pXO2⁻), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture. PMID:21827967

  5. Optimizing PHB and Protease Production by Box Behnken Design

    Amro Abd al fattah Amara

    2013-04-01

    Full Text Available Mixed culture is more suitable to adapt more flexible fermentation process and produce different product simultaneously. In this study a mixed Bacillus culture was investigated for their ability to produce the bioplastic "Polyhydroxybutyrate" and both of the mesophilic and the thermophilic proteases in one flask. Box-Behnken experimental design was used. The produced amount of PHB has been increased significantly. Meanwhile there is a competition between PHB and proteases. The maximum produced amount of PHB using Box-Behnken design was 2.82 g/l/48 h with protease activity equal to 41.9 Units/ml/48 h for thermophilic proteases and 99.65 Units/ml/48 h for mesophilic proteases. Excel solver was used for extra-optimization for the optimum conditions obtained from Box-Behnken experiments and its model. The maximum PHB obtained after using Excel solver was 2.88 g/l/48 h. The maximum mesophilic and thermophilic activities obtained at the same PHB production conditions were 175.68 and 243.38 Units/ml respectively. The model accuracy as obtained from Excel solver was 118.8%, which prove the power of the experimental design in optimizing such complicated process. The strategies used in this study are recommended for the production of PHB and different proteases simultaneously using Bacillus mixed culture. ABSTRAK: Kultur campuran adalah lebih sesuai bagi proses penapaian yang fleksibel dan ia boleh menghasilkan produk yang berbeza secara serentak. Dalam kajian ini keupayaan  menghasilkan "Polyhydroxybutyrate" bioplastik serta mesofilik dan termofilik protease dalam satu flask oleh  kultur Bacillus campuran telah disiasat. Eksperimen rekabentuk Box-Behnken telah digunakan. Jumlah PHB yang dikeluarkan meningkat dengan ketara dan terdapat persaingan antara PHB dan protease. Jumlah keluaran PHB maksima menggunakan rekabentuk Box-Behnken adalah 2.82 g/l/48 jam dengan aktiviti protease sama dengan 41.9 Unit/ml/48 jam untuk protease termofilik dan 99.65 Unit

  6. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    Lilburn Timothy G

    2011-12-01

    Full Text Available Abstract Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome in the malaria parasite Plasmodium falciparum and its sibling species 123, providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database 4, and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H system 5, blood stage microarray experiments 678, proteomics 9101112, literature text mining, and sequence homology analysis. Seventy-seven (77 out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs. These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins, range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide

  7. OPTIMIZATION OF PROTEASE PRODUCTION FROM FUNGI ISOLATED FROM SOIL

    Sonia Sethi

    2015-07-01

    Full Text Available Fungal strains isolated from soil by serial dilution method were screened for alkaline protease production. Isolate Penicillium chrysogenum the most potent producer of alkaline protease was identified. The isolate showed highest activity in the optimized medium at pH 9.0, temperature 35ºC, with 1% soycake and peptone incubated for 7 days. Proteases represent one of the largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes.

  8. Hyper production of alkaline protease by mutagenized bacillus subtilis

    The purpose of this work was to augment the alkaline protease production from Bacillus subtilis by using chemical mutagen (MMS) and UV mutagenesis. A number of mutants were isolated which produce high levels of extra cellular proteases. Analysis of culture supernatants of these mutants had shown that the total amounts of proteolysis activity were increased from 1 to 2 fold over the wild strain. Clones showing promote response were further characterized by analyzing different parameters; like of Temperature, pH substrate concentration and incubation period, to study the activity of protease enzyme. (author)

  9. Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis

    Skjødt, Mikkel-Ole; Palarasah, Yaseelan; Rasmussen, Karina Juhl;

    2010-01-01

    The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intest...... protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway....

  10. The structural basis for catalysis and substrate specificity of a rhomboid protease

    Vinothkumar, Kutti R.; Strisovsky, Kvido; Andreeva, Antonina; Christova, Yonka; Verhelst, Steven; Freeman, Matthew

    2010-01-01

    While soluble proteases are some of the best-studied enzymes, this first structure of a rhomboid protease bound to a mechanism-based inhibitor provides unprecedented functional insights into the recently discovered class of intramembrane proteases, illustrating both similarities and differences with classical serine proteases.

  11. Protease inhibitor expression in soybean roots exhibiting susceptible and resistance reactions to soybean cyst nematode

    Protease inhibitors play a role in regulating proteases during cellular development and in plant defense against insects and nematodes. We identified, cloned and sequenced cDNAs encoding six protease inhibitors expressed in soybean roots infected with soybean cyst nematode. Four of these protease in...

  12. Targeting Proteases in Cardiovascular Diseases by Mass Spectrometry-Based Proteomics

    Klingler, Diana; Hardt, Markus

    2012-01-01

    Proteases hydrolyze peptide bonds, thereby controlling the function of proteins and peptides on the posttranslational level. In the cardiovascular system, proteases play pivotal roles in the regulation of blood pressure, coagulation and other essential physiological processes. Accordingly, proteases are prime targets for therapeutic interventions and diagnostics. Proteases are part of complex proteolytic networks comprised of enzymes, inhibitors, activators, substrates and cleavage products. ...

  13. Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease.

    Su, Shih-Chieh; Lin, Chien-Chu; Tai, Hui-Chung; Chang, Mu-Yueh; Ho, Meng-Ru; Babu, C Satheesan; Liao, Jiahn-Haur; Wu, Shih-Hsiung; Chang, Yuan-Chih; Lim, Carmay; Chang, Chung-I

    2016-05-01

    The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85 Å crystal structure of Mg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA. PMID:27041593

  14. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    Bazan, J F; Fletterick, R J

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserv...

  15. Cysteine protease cathepsins and matrix metalloproteinases in the development of abdominal aortic aneurysms

    Qin, Yanwen; Cao, Xu; Yang, Yaoguo; Shi, Guo-Ping

    2013-01-01

    Both cysteine protease cathepsins and matrix metalloproteinases are implicated in the pathogenesis of abdominal aortic aneurysms (AAAs) in humans and animals. Blood and aortic tissues from humans or animals with AAAs contain much higher levels of these proteases, and often lower levels of their endogenous inhibitors, than do blood and aortic tissues from healthy subjects. Protease- and protease inhibitor-deficient mice and synthetic protease inhibitors have affirmed that cysteinyl cathepsins ...

  16. Partial Purification and Characterization of a Cysteine Protease Inhibitor from the Plerocercoid of Spirometra erinacei

    Chung, Young-Bae; Yang, Hyun-Jong

    2008-01-01

    Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargan...

  17. The Place of protease inhibitors in antiretroviral treatment

    S.B. Tenore

    2009-10-01

    Full Text Available With the introduction of highly active antiretroviral therapy, a number of drugs have been developed. The best choice concerning which antiretroviral analogs to start is always under discussion, especially in the choice between non-nucleoside reverse transcriptase inhibitors-based therapies and ritonavir-boosted protease inhibitors. Both are proven to control viral replication and lead to immunological gain. The choice between a non-nucleoside analog reverse transcriptase inhibitor and a protease inhibitor as a third antiretroviral drug in the therapy should consider factors related to the individual, as well as the inclusion of the best therapy in the patient's daily activities and potential adherence. The protease inhibitor-based therapies showed similar efficacy among the various inhibitors with characteristics concerning the adverse events from each medicine. For the treatment of protease-resistant patients, darunavir and tipranavir showed good efficacy with higher genetic barrier to resistance.

  18. Improving Viral Protease Inhibitors to Counter Drug Resistance.

    Kurt Yilmaz, Nese; Swanstrom, Ronald; Schiffer, Celia A

    2016-07-01

    Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design. PMID:27090931

  19. Effect of Gastrointestinal Protease Digestion on Bioactivity of Marine Peptides

    Jensen, Ida-Johanne; Andersen, Lisa Lystbæk; Ossum, Carlo Gunnar; Jakobsen, Greta; De Gobba, Christian; Farvin, Sabeena; Johansson, Inez; Hoffmann, Else; Oddny Elvevoll, Edel; Jessen, Flemming; Nielsen, Henrik Hauch

    2014-01-01

    executed without concerning subsequent digestion after intake and the aim of this work was hence to investigate how the in vitro antioxidative, antihypertensive and caspase activating activities of peptides are affected by digestion with gastrointestinal (GI) proteases. Five different fish protein...... hydrolysates were chosen to study the effect of in vitro digestion on bioactivity. The protein concentration decreased in all samples during digestion and the molecular weight distribution of the peptides shifted towards lower values. Thus, in vitro digestion with GI proteases resulted in a further degradation...... of the peptides obtained by hydrolysis. The antihypertensive effect increased in all samples after digestion with GI proteases whereas the antioxidative capacity decreased. The effect on the caspase activity depended on the proteases used in the preparation of hydrolysates. In conclusion, the caspase...

  20. The maize cystatin CC9 interacts with apoplastic cysteine proteases

    van der Linde, Karina; Mueller, André N.; Hemetsberger, Christoph; Kashani, Farnusch; Van der Hoorn, Renier A. L.; Doehlemann, Gunther

    2012-01-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signa...

  1. Salivary protease inhibitors with non anti-hemostatic function

    Chmelař, Jindřich; Francischetti, I.M.B.; Kotsyfakis, Michalis

    Dordrecht : Springer Science+Business Media, 2010 - (Kini, R.; Clemetson, K.; Markland, F.; McLane, M.; Morita, T.), s. 153-164 ISBN 978-90-481-9294-6 R&D Projects: GA AV ČR IAA600960811 Institutional research plan: CEZ:AV0Z60220518 Keywords : Cysteine protease inhibitors * Serine protease inhibitors * Arthropod saliva * Immunity * Inflammation * Cystatin * Tryptase * Elastase * Kunitz domain Subject RIV: EC - Immunology

  2. The non-death role of metacaspase proteases

    AmitShrestha

    2012-01-01

    The activation of caspase proteases and the targeting of protein substrates act as key steps in the engagement and conduct of apoptosis/programmed cell death. However, the discovery of caspase involvement in diverse non-apoptotic cellular functions strongly suggests that these proteins may have evolved from a core behaviour unrelated to the induction of cell death. The presence of similar proteases, termed metacaspases, in single cell organisms supports the contention that such proteins may h...

  3. Purification and characterization of alkaline protease from Lysinibacillus fusiformis

    Suppiah S*; Sendeshkannan K; Prabakaran P; Rajkumar G; Yasothkumar N

    2012-01-01

    A novel alkaline protease producing bacterium was isolated from the gut of an estuarine fish Etroplus suratensis. The strain was identified by sequencing the fragment of their bacterial 16s rRNA and its homology was 97% closest to the Lysinibacillus fusiformis. An extracellular protease from this organism was purified by acetone precipitation, ion exchange chromatography and gel filtration chromatography methods and the specific activity of the purified enzyme was found to be 20.39 U/mg, 169....

  4. ADAM 12 protease induces adipogenesis in transgenic mice

    Kawaguchi, Nobuko; Xu, Xiufeng; Tajima, Rie; Kronqvist, Pauliina; Sundberg, Christina; Loechel, Frosty; Albrechtsen, Reidar; Wewer, Ulla M

    2002-01-01

    ADAM 12 (meltrin-alpha) is a member of the ADAM (a disintegrin and metalloprotease) family. ADAM 12 functions as an active metalloprotease, supports cell adhesion, and has been implicated in myoblast differentiation and fusion. Human ADAM 12 exists in two forms: the prototype membrane-anchored pr...... adipogenic phenotype, suggesting a requirement for ADAM 12 protease activity. This is the first in vivo demonstration that an ADAM protease is involved in adipogenesis....

  5. Amplified detection of protease activity using porous silicon nanostructures

    Orosco, Manuel

    This dissertation will focus on harnessing the optical properties of porous silicon to sense protease activity. Electrochemical etching of polished silicon wafers produces porous silicon with unique optical properties such as Fabry-Perot fringes or a dielectric mirror reflecting specific wavelengths. Porous silicon optical transducers are coupled to a biochemical reaction (protease activity) and optically measured in a label-free manner. The first chapter is an introductory chapter discussing the current methods of detecting protease activity. Also discussed is the use of porous silicon for label-free sensing. The second chapter discusses the use of thin protein layers that are spin coated on the surface of a porous silicon film and excluded from the porous matrix based on size. When active proteases are introduced to the protein layer, small peptide fragments are generated, causing a change in refractive index from low to high. This can be used as a tool to monitor protease activity and amplify the signal to the naked eye. To extend on the second chapter, a double layered porous silicon film with the first layer have large pores and the second layer etched below having small pores was used for sensing protease activity. Proteases are adsorbed into the first layer and introduction of whole protein substrate produces small peptide fragments that can enter the second layer (changing the effective optical thickness). The fourth chapter describes a method of using luminescent transducers coupled to protein films. An "on-off" sensor using protein coated luminescent porous silicon was used to detect a decrease in the intensity of luminescence due to degradation of the protein film. An "off-on" sensor involved a fluorescent dye housed in the porous film and capped with a protein coating. The release of the dye is caused by the action of a protease causing an increase in fluorescent intensity from the dye.

  6. Identification of an Archaeal Presenilin-Like Intramembrane Protease

    Torres-Arancivia, Celia; Ross, Carolyn M.; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

    2010-01-01

    Background The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demo...

  7. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  8. Characterizing Protease Specificity: How Many Substrates Do We Need?

    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  9. Effect of protease inhibitors on the sense of taste.

    Schiffman, S S; Zervakis, J; Heffron, S; Heald, A E

    1999-10-01

    The purpose of this study was to investigate the taste properties of protease inhibitors which are essential components of drug regimes used to treat human immunodeficiency virus (HIV) infection. In this study, the taste properties of four protease inhibitors (indinavir, ritonavir, saquinavir, and nelfinavir) were investigated in unmedicated HIV-infected patients and healthy controls. Three of the four protease inhibitors (indinavir, ritonavir, and saquinavir) were found to be predominantly bitter (with additional qualities of medicinal, metallic, astringent, sour, and burning). Nelfinavir was found to be relatively tasteless. HIV-infected and uninfected control subjects detected protease inhibitors at similar concentrations, but HIV-infected subjects perceived suprathreshold concentrations as more bitter than controls. Detection thresholds ranged from 0.0061 mM for saquinavir in HIV-infected patients to 0.0702 mM for ritonavir in uninfected control subjects. Suprathreshold studies indicated that protease inhibitors modified the taste perception of a variety of other taste compounds. These results are consistent with clinical findings that protease inhibitors produce taste complaints that can impact patient compliance. PMID:10501290

  10. Protease inhibitors decrease the resistance of Vitaceae to Plasmopara viticola.

    Gindro, Katia; Berger, Valentine; Godard, Sophie; Voinesco, Francine; Schnee, Sylvain; Viret, Olivier; Alonso-Villaverde, Virginia

    2012-11-01

    Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll). PMID:22906813

  11. Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

    Eiichi Saitoh; Shinya Yamamoto; Eishiro Okamoto; Yoshimi Hayakawa; Takashi Hoshino; Ritsuko Sato; Satoko Isemura; Sadami Ohtsubo; Masayuki Taniguchi

    2007-01-01

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic ...

  12. Protease encoding microbial communities and protease activity of the rhizosphere and bulk soils of two maize lines with different N uptake efficiency.

    Baraniya, Divyashri; Puglisi, Edoardo; Ceccherini, Maria Teresa; Pietramellara, Giacomo; Giagnoni, Laura; Arenella, Mariarita; Nannipieri, Paolo; Renella, Giancarlo

    2016-01-01

    This study was carried out to understand the interplay of plant Nitrogen Utilizing Efficiency (NEU) with protease activtiy and microbial proteolytic community composition in the rhizosphere and bulk soils. Protease activity, diversity and abundance of protease genes (using DGGE and qPCR respectively of two key bacterial protease encoding genes: alkaline metallo-peptidase (apr) and neutral-metallopeptidases (npr) were monitored in both rhizosphere and bulk soils from two maize in-bred lines L0...

  13. Secretory Granule Proteases in Rat Mast Cells. Cloning of 10 Different Serine Proteases and a Carboxypeptidase A from Various Rat Mast Cell Populations

    Lützelschwab, Claudia; Pejler, Gunnar; Aveskogh, Maria; Hellman, Lars

    1997-01-01

    Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the...

  14. Modelling of potentially promising SARS protease inhibitors

    Plewczynski, Dariusz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Hoffmann, Marcin [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Grotthuss, Marcin von [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Knizewski, Lukasz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Rychewski, Leszek [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Eitner, Krystian [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Ginalski, Krzysztof [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland)

    2007-07-18

    In many cases, at the beginning of a high throughput screening experiment some information about active molecules is already available. Active compounds (such as substrate analogues, natural products and inhibitors of related proteins) are often identified in low throughput validation studies on a biochemical target. Sometimes the additional structural information is also available from crystallographic studies on protein and ligand complexes. In addition, the structural or sequence similarity of various protein targets yields a novel possibility for drug discovery. Co-crystallized compounds from homologous proteins can be used to design leads for a new target without co-crystallized ligands. In this paper we evaluate how far such an approach can be used in a real drug campaign, with severe acute respiratory syndrome (SARS) coronavirus providing an example. Our method is able to construct small molecules as plausible inhibitors solely on the basis of the set of ligands from crystallized complexes of a protein target, and other proteins from its structurally homologous family. The accuracy and sensitivity of the method are estimated here by the subsequent use of an electronic high throughput screening flexible docking algorithm. The best performing ligands are then used for a very restrictive similarity search for potential inhibitors of the SARS protease within the million compounds from the Ligand.Info small molecule meta-database. The selected molecules can be passed on for further experimental validation.

  15. The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

    Ruth M Kennan

    Full Text Available Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by

  16. Computational analysis of HIV-1 protease protein binding pockets.

    Ko, Gene M; Reddy, A Srinivas; Kumar, Sunil; Bailey, Barbara A; Garg, Rajni

    2010-10-25

    Mutations that arise in HIV-1 protease after exposure to various HIV-1 protease inhibitors have proved to be a difficult aspect in the treatment of HIV. Mutations in the binding pocket of the protease can prevent the protease inhibitor from binding to the protein effectively. In the present study, the crystal structures of 68 HIV-1 proteases complexed with one of the nine FDA approved protease inhibitors from the Protein Data Bank (PDB) were analyzed by (a) identifying the mutational changes with the aid of a developed mutation map and (b) correlating the structure of the binding pockets with the complexed inhibitors. The mutations of each crystal structure were identified by comparing the amino acid sequence of each structure against the HIV-1 wild-type strain HXB2. These mutations were visually presented in the form of a mutation map to analyze mutation patterns corresponding to each protease inhibitor. The crystal structure mutation patterns of each inhibitor (in vitro) were compared against the mutation patterns observed in in vivo data. The in vitro mutation patterns were found to be representative of most of the major in vivo mutations. We then performed a data mining analysis of the binding pockets from each crystal structure in terms of their chemical descriptors to identify important structural features of the HIV-1 protease protein with respect to the binding conformation of the HIV-1 protease inhibitors. Data mining analysis is performed using several classification techniques: Random Forest (RF), linear discriminant analysis (LDA), and logistic regression (LR). We developed two hybrid models, RF-LDA and RF-LR. Random Forest is used as a feature selection proxy, reducing the descriptor space to a few of the most relevant descriptors determined by the classifier. These descriptors are then used to develop the subsequent LDA, LR, and hierarchical classification models. Clustering analysis of the binding pockets using the selected descriptors used to

  17. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease. PMID:22888800

  18. The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

    Prasad, Lata; Leduc, Yvonne; Hayakawa, Koto; Delbaere, Louis T.J. (Saskatchewan)

    2008-06-27

    V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

  19. Targeting exosites on blood coagulation proteases

    Robson Q. Monteiro

    2005-06-01

    Full Text Available The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa. Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa. We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os

  20. Modeling and structural analysis of PA clan serine proteases

    Laskar Aparna

    2012-05-01

    Full Text Available Abstract Background Serine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques. Results Our predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core

  1. Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy.

    Giandhari, Jennifer; Basson, Adriaan E; Sutherland, Katherine; Parry, Chris M; Cane, Patricia A; Coovadia, Ashraf; Kuhn, Louise; Hunt, Gillian; Morris, Lynn

    2016-04-01

    Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of

  2. Mitochondrial cereblon functions as a Lon-type protease

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria. PMID:27417535

  3. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A. (UMASS, MED)

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  4. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    Verma, Amit; Ansari, Mohammad W; Anwar, Mohmmad S; Agrawal, Ruchi; Agrawal, Sanjeev

    2014-05-01

    Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films. PMID:24122212

  5. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles. PMID:26955746

  6. Current trends and challenges in proteomic identification of protease substrates.

    Vizovišek, Matej; Vidmar, Robert; Fonović, Marko; Turk, Boris

    2016-03-01

    Proteolytic cleavage is a ubiquitous, irreversible, posttranslational modification that changes protein structure and function and plays an important role in numerous physiological and pathological processes. Over the last decade, proteases have become increasingly important clinical targets because many of their inhibitors are already used in the clinic or in various stages of clinical testing. Therefore, a better understanding of protease action and their repertoires of physiological substrates can not only provide an important insight into their mechanisms of action but also open a path toward novel drug design. Historically, proteases and their substrates were mainly studied on a case-by-case basis, but recent advancements in mass spectrometry-based proteomics have enabled proteolysis studies on a global scale. Because there are many different types of proteases that can operate in various cellular contexts, multiple experimental approaches for their degradomic characterization had to be developed. The present paper reviews the mass spectrometry-based approaches for determining the proteolytic events in complex biological samples. The methodologies for substrate identification and the determination of protease specificity are discussed, with a special focus on terminomic strategies, which combine peptide labeling and enrichment. PMID:26514758

  7. Characterization of two uterine proteases and their actions on the estrogen receptor

    We have characterized two previously undetected proteases from the calf uterine cytosol and measured their actions on the estrogen receptor. One is an exopeptidase, purified 60-fold, that hydrolyzed amino acid (lysine-, and alanine-, or leucine-) p-nitroanilide substrates and leucyl-glycylglycine, did not hydrolyze [14C]methemoglobin, was completely inhibited by 1 mM bestatin or puromycin (specific inhibitors of leucine aminopeptidase like enzymes), and was unable to influence the sedimentation of the 8S form of the estrogen receptor in sucrose gradients containing dilute Tris buffer. A commercial porcine leucine aminopeptidase, like the calf uterine aminopeptidase, did not convert the 8S estrogen receptor to a 4S form. Evidently, removal of the N-terminal amino acid(s) from the estrogen receptor by exopeptidase action cannot alter the sedimentation of the 8S form of the receptor, or the N-terminal amino acid(s) of the receptor is (are) unaccessible or resistant to exopeptidase activity. The second, a receptor-active protease, is an endopeptidase that did not hydrolyze any of the synthetic amide or peptide substrates tested but did possess [14C]methemoglobin-degrading activity and the ability to convert the 8S estrogen receptor to a modified 4S form in sucrose gradients containing dilute Tris buffer. The modified 4S receptor was separable from the native receptor by DEAE-cellulose chromatography. The endopeptidase did not require Ca2+ for activity, and its chromatographic properties were distinctly different from a previously isolated Ca2+-activated protease. It was inhibited by leupeptin or dipyridyl disulfide, suggesting the presence of a thiol group that is essential for its activity

  8. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  9. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    Marian Dorcas Quain

    2013-08-01

    Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

  10. Screening of Alkaline Protease-Producing Streptomyces diastaticus and Optimization of Enzyme Production

    Elham Dawoodi

    2014-12-01

    Full Text Available Background and Aim: Alkaline proteases are used in pharmaceutical, film and photography, silk production and food, leather and detergent industries. Actinomycetes are gram positive bacteria that produce different enzymes such as proteases. The aims of this research were isolation of native alkaline protease-producing Actinomycete spp. from different soil samples as well as optimizing the conditions for enzyme production. Materials and Methods: The different soil samples were collected from different locations of the provinces of Khouzestan, Chahar Mahalo Bakhtiari and Isfahan, Iran. After determining of the best alkaline protease producing species using Lowry method, the optimization of alkaline protease was performed. Results: The alkaline protease producing Actinomycete spp. was isolated from soil. The most enzyme activity was measured in S.diastaticus. The best concentration of sucrose as the carbon source for the highest production of alkaline protease was 10 g/l. The optimum pH and temperature for the alkaline protease production by S. diastaticus were 10 and 30°C respectively. The maximum activity of alkaline protease was measured at 200 rpm as the best aeration speed. Conclusions: This is the first report of alkaline protease production by Streptomyces diastaticus in Iran. The accomplished examinations in this research confirmed the previous theories of alkaline protease production by Actinomycetes relatively. Regarding the immense applications of alkaline proteases in several industries and isolation of a native alkaline protease producing Actinomycete, The production potential of this enzyme in our country could be accessible in the near future.

  11. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant–pathogen interactions

    Jashni, Mansoor Karimi; Mehrabi, Rahim; Collemare, Jérôme; Mesarich, Carl H.; de Wit, Pierre J. G. M.

    2015-01-01

    Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered i...

  12. Lvserpin3 is involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

    Liu, Yongjie; Liu, Tao; Hou, Fujun; Wang, Xianzong; Liu, Xiaolin

    2016-01-01

    Serine protease inhibitor, represented by serpin, plays an important inhibitory role on proteases involved in the immune responses. To clarify the immune characterizations of serpin, a novel serpin (Lvserpin3) encoding for 410 amino acids with a 23-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Lvserpin3 expressed strongest in hepatopancreas, and was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Suppression of Lvserpin3 by dsRNA led to a significant increase in the transcripts of LvPPAF, LvproPO and phenoloxidase (PO) activity, and also led to the high cumulative mortality. The recombinant Lvserpin3 protein (rLvserpin3) inhibited the proteases secreted by M. lysodeikticus and Bacillus subtilis, and further exhibited inhibitory role on the growth of B. subtilis and M. lysodeikticu. Moreover, rLvserpin3 was found to be able to block the activation of prophenoloxidase system. Taken together, the results imply that Lvserpin3 may be involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system. PMID:26432049

  13. An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

    Piero Giansanti

    2015-06-01

    Full Text Available Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti4+-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS. For specific sites, this approach increases their detectability by more than 1,000-fold.

  14. Substrate specificity of the ubiquitin and Ubl proteases

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  15. Protease-specific nanosensors for magnetic resonance imaging.

    Schellenberger, Eyk; Rudloff, Franziska; Warmuth, Carsten; Taupitz, Matthias; Hamm, Bernd; Schnorr, Jörg

    2008-12-01

    Imaging of enzyme activity is a central goal of molecular imaging. With the introduction of fluorescent smart probes, optical imaging has become the modality of choice for experimental in vivo detection of enzyme activity. Here, we present a novel high-relaxivity nanosensor that is suitable for in vivo imaging of protease activity by magnetic resonance imaging. Upon specific protease cleavage, the nanoparticles rapidly switch from a stable low-relaxivity stealth state to become adhesive, aggregating high-relaxivity particles. To demonstrate the principle, we chose a cleavage motif of matrix metalloproteinase 9 (MMP-9), an enzyme important in inflammation, atherosclerosis, tumor progression, and many other diseases with alterations of the extracellular matrix. On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles (PSOP) have a hydrodynamic diameter of only 25 nm. PSOP are rapidly activated, resulting in aggregation and increased T2*-relaxivity. PMID:19007261

  16. The binding mechanism of a peptidic cyclic serine protease inhibitor

    Jiang, Longguang; Svane, Anna S P; Sørensen, Hans Peter; Jensen, Jan K; Hosseini, Masood; Chen, Zhuo; Weydert, Caroline; Nielsen, Jakob T; Christensen, Anni; Yuan, Cai; Jensen, Knud Jørgen; Nielsen, Niels Chr; Malmendal, Anders; Huang, Mingdong; Andreasen, Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  17. HIV-1 Protease Mutations and Protease Inhibitor Cross-Resistance▿ † ‡

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W. Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W.

    2010-01-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  18. Schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor.

    Maha-Hamadien Abdulla

    2007-01-01

    Full Text Available BACKGROUND: Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization-recommended drug, but concerns over drug resistance encourage the search for new drug leads. METHODS AND FINDINGS: The efficacy of the vinyl sulfone cysteine protease inhibitor K11777 was tested in the murine model of schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology including hepato- and splenomegaly, presence of parasite egg-induced granulomas in the liver, and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25 mg/kg twice daily [BID], administered intraperitoneally at the time of parasite migration through the skin and lungs (days 1-14 postinfection [p.i.], resulted in parasitologic cure (elimination of parasite eggs in five of seven cases and a resolution of other disease parameters. K11777 (50 mg/kg BID, administered at the commencement of egg-laying by mature parasites (days 30-37 p.i., reduced worm and egg burdens, and ameliorated organ pathology. Using protease class-specific substrates and active-site labeling, one molecular target of K11777 was identified as the gut-associated cathepsin B1 cysteine protease, although other cysteine protease targets are not excluded. In rodents, dogs, and primates, K11777 is nonmutagenic with satisfactory safety and pharmacokinetic profiles. CONCLUSIONS: The significant reduction in parasite burden and pathology by this vinyl sulfone cysteine protease inhibitor validates schistosome cysteine proteases as drug targets and offers the potential of a new direction for chemotherapy of human schistosomiasis.

  19. Purification and characterization of alkaline proteases from aspergillus terreus

    Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermo stable and included in the category of alkaline serine and metallo protease. During partial purification, presence of enzyme in 60% (NH/sub 4/)/sub 2/SO/sub 4/ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermo stable towards high temperature (60 deg. C), however it denatured irreversibly at 70 deg. C showing 80% loss of activity. The maximum proteolytic activity was found at 40 deg. C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg/sup +2/, Mn/sup +2/ and Fe/sup +3/ (1 mM each) showed minute stimulatory effects on enzyme activity. Co/sup +2/ and Ca/sup +2/ (1 mM) had neither excitatory nor inhibitory effect while Hg/sup +2/ and Cu/sup +2/ (1 mM) slightly reduced the enzyme activity. (author)

  20. Trichuris suis: thiol protease activity from adult worms.

    Hill, D E; Sakanari, J A

    1997-01-01

    Trichuris suis, the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adult T. suis and from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mM dithiothreitol or 5 mM cysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes. PMID:9024202

  1. Conformational Stability of Hepatitis C Virus NS3 Protease

    Abian, Olga; Vega, Sonia; Neira, Jose Luis; Velazquez-Campoy, Adrian

    2010-01-01

    The hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role....

  2. Design of HIV protease inhibitors based on inorganic polyhedral metallacarboranes

    Řezáčová, Pavlína; Pokorná, Jana; Brynda, Jiří; Kožíšek, Milan; Cígler, Petr; Lepšík, Martin; Fanfrlík, Jindřich; Řezáč, Jan; Grantz Šašková, Klára; Sieglová, Irena; Plešek, Jaromír; Šícha, Václav; Grüner, Bohumír; Oberwinkler, H.; Sedláček, Juraj; Kräusslich, H. G.; Hobza, Pavel; Král, V.; Konvalinka, Jan

    2009-01-01

    Roč. 52, č. 22 (2009), s. 7132-7141. ISSN 0022-2623 R&D Projects: GA AV ČR IAAX00320901; GA MŠk LC512; GA MŠk LC523 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514; CEZ:AV0Z40320502 Keywords : HIV protease inhibitors * aspartic proteases * viral resistance * cobalt bis(dicarbollide) * crystal structure Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2009

  3. Expression and secretion of heterologous proteases by Corynebacterium glutamicum.

    Billman-Jacobe, H; Wang, L.; Kortt, A; Stewart, D; Radford, A

    1995-01-01

    Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding s...

  4. CLIP proteases and Plasmodium melanization in Anopheles gambiae.

    Barillas-Mury, Carolina

    2007-07-01

    Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium. PMID:17512801

  5. ISOLASI DAN KARAKTERISASI PROTEASE DARI BAKTERI TANAH RAWA INDRALAYA, SUMATERA SELATAN [Isolation and Characterization of Proteases from Indralaya Soil Swamp Bacteria,South Sumatera

    Ace Baehaki*

    2011-06-01

    Full Text Available In an effort of obtaining indigenous protease producing bacteria, screening for bacterial protease was conducted from samples collected from Indralaya soil swamp, South Sumatera. Three of 31 colonies showed high protease activity with proteolytic index >1.00. T1S1 produced enzyme with the highest activity. The crude enzyme activity after 48 hours of incubation was 0.391 IU/ml. The optimum pH of the extracelull proteases from T1S1, T3S2 and T3S3 were 8.0, 8.0, and 7.5, respectively. The optimum temperature of T1S1, T3S2 and T3S3 proteases were 40, 50, and 500C, respectively. All metal ions tested (Na+, K+, Mn2+, Zn2+ and Fe2+ inhibited proteases except Fe2+ which activatesthe T3S3 protease at 5 mM. EDTA (1 and 5 mM inhibited all proteases. Study on the effect of metals ion and spesific inhibitors indicated that all protease are metaloprotease. Molecular weights was determined using SDS-PAGE and zymogram technique. The molecular weight of T1S1 proteases was 121 kD,T3S2 proteaseswere 51, 71, and 119 kD whereas T3S3 proteaseswere 49, 70, and 116 kD.

  6. Genetically Engineered Mouse Models Reveal the Importance of Proteases as Drug Targets in Osteoarthritis

    Miller, Rachel E; Lu, Yongzhi; Tortorella, Micky D.; Malfait, Anne-Marie

    2013-01-01

    More than two decades of research has revealed a network of proteases that orchestrates cartilage degradation in osteoarthritis. This network includes not only metalloproteinases that degrade the major macromolecules in cartilage, aggrecan and type II collagen, but also serine proteases and cysteine proteases, such as cathepsin K. The current review summarizes the role of proteases in osteoarthritis progression, based on studies in genetically engineered mouse models. In addition, a brief ove...

  7. Induction of exocellular proteases from N. crassa under conditions of nitrogen starvation

    Protease induction from N. crassa starved for nitrogen was found to be similar in requirements to protease induction for carbon-starved cells. The primary difference between the two processes is that the nitrogen-starved cells do not require added protease for induction; however, concentration effects for protein substrate, nature of amino acid repression of the process, and kinetics of protease synthesis were almost identical in the two systems. (U.S.)

  8. Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

    Mourão, Caroline B.F.; Elisabeth F Schwartz

    2013-01-01

    The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomo...

  9. Modulation of visceral pain and inflammation by protease-activated receptors

    Vergnolle, Nathalie

    2004-01-01

    The gastrointestinal (GI) tract is exposed to a large array of proteases, under both physiological and pathophysiological conditions. The discovery of G protein-coupled receptors activated by proteases, the protease-activated receptors (PARs), has highlighted new signaling functions for proteases in the GI tract, particularly in the domains of inflammation and pain mechanisms. Activation of PARs by selective peptidic agonists in the intestine or the pancreas leads to inflammatory events and c...

  10. Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

    Carroll, Ian M.; Ringel-Kulka, Tamar; Ferrier, Laurent; Wu, Michael C.; Siddle, Jennica P.; Bueno, Lionel; Ringel, Yehuda

    2013-01-01

    Objective: Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear. Aim: The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples. Design: In order to capture a wide range of fec...

  11. Semi-continuous in situ magnetic separation for enhanced extracellular protease productionmodeling and experimental validation

    Cerff, M.; Scholz, A.; Käppler, T.;

    2013-01-01

    In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constit...... production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc....

  12. Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli.

    Zhao, Mingzhi; Cai, Man; Wu, Feilin; Zhang, Yao; Xiong, Zhi; Xu, Ping

    2016-10-01

    Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product. PMID:27260967

  13. Analysis of regulatory protease sequences identified through bioinformatic data mining of the Schistosoma mansoni genome

    Minchella Dennis J

    2009-10-01

    Full Text Available Abstract Background New chemotherapeutic agents against Schistosoma mansoni, an etiological agent of human schistosomiasis, are a priority due to the emerging drug resistance and the inability of current drug treatments to prevent reinfection. Proteases have been under scrutiny as targets of immunological or chemotherapeutic anti-Schistosoma agents because of their vital role in many stages of the parasitic life cycle. Function has been established for only a handful of identified S. mansoni proteases, and the vast majority of these are the digestive proteases; very few of the conserved classes of regulatory proteases have been identified from Schistosoma species, despite their vital role in numerous cellular processes. To that end, we identified protease protein coding genes from the S. mansoni genome project and EST library. Results We identified 255 protease sequences from five catalytic classes using predicted proteins of the S. mansoni genome. The vast majority of these show significant similarity to proteins in KEGG and the Conserved Domain Database. Proteases include calpains, caspases, cytosolic and mitochondrial signal peptidases, proteases that interact with ubiquitin and ubiquitin-like molecules, and proteases that perform regulated intramembrane proteolysis. Comparative analysis of classes of important regulatory proteases find conserved active site domains, and where appropriate, signal peptides and transmembrane helices. Phylogenetic analysis provides support for inferring functional divergence among regulatory aspartic, cysteine, and serine proteases. Conclusion Numerous proteases are identified for the first time in S. mansoni. We characterized important regulatory proteases and focus analysis on these proteases to complement the growing knowledge base of digestive proteases. This work provides a foundation for expanding knowledge of proteases in Schistosoma species and examining their diverse function and potential as targets

  14. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus

    Mothe, Thirumala; Sultanpuram, Vishnuvardhan Reddy

    2016-01-01

    Alkaline proteases are important enzymes in many industrial applications, especially as additives in laundry detergent industry. Though there are a number of Bacillus species which are reported to be producing proteases, the efficiency of a protease produced by a novel strain has to be studied in comparison to the others. Hence, in this study, an alkaline serine protease produced by a novel species Bacillus caseinilyticus was purified and characterized for its possible usage in detergent indu...

  15. Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

    RATIA, Kiira; Saikatendu, Kumar Singh; Bernard D. Santarsiero; Barretto, Naina; Baker, Susan C.; Stevens, Raymond C.; MESECAR, Andrew D.

    2006-01-01

    Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-Å crystal structure of the catalytic core of ...

  16. SjAPI, the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

    Chen, Zongyun; Wang, Bin; Hu, Jun; Yang, Weishan; Cao,Zhijian; Zhuo, Renxi; Li, Wenxin; Wu, Yingliang

    2013-01-01

    Background Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. Principal Findings Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin ...

  17. Boosted protease inhibitors and the electrocardiographic measures of QT and PR durations

    Soliman, Elsayed Z; Lundgren, Jens D; Roediger, Mollie P;

    2011-01-01

    There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown.......There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown....

  18. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    Fabíola Dorneles Inácio; Roselene Oliveira Ferreira; Caroline Aparecida Vaz de Araujo; Tatiane Brugnari; Rafael Castoldi; Rosane Marina Peralta; Cristina Giatti Marques de Souza

    2015-01-01

    Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good p...

  19. PENGEMPUKAN DAGING DENGAN ENZIM PROTEASE TANAMAN BIDURI (Calotropis gigantea [Meat Tenderization using Protease of Biduri Plant (Calotropis gigantea

    Erni Sofia Murtini1

    2003-12-01

    Full Text Available Tenderness is the main attribute quality of meat, which influences consumer acceptability. Protease enzyme (like papain, bromelin and ficin are known to be used for improving tenderness of meat trough degradation of the protein. Biduri plant (Calotropis gigantea contains protease enzyme in its latex or the young tissue (0-20 cm plant tip. After isolation of crude enzyme using ammonium sulphate, the enzyme was the applied to tenderise meat at concentrations 0 ; 0,25; 0,5; 0,75 and 1,0%. The result showed that concentration of protease enzyme affected to meat tenderness that determined by compression test and tensile strength. The enzyme (0.5% was enough to tenderise meat indicated by decreasing its compression test value to 201,160 N 9 from control of 228,582 N and tensile strength value to 4,618 N (from control 9,588N

  20. Protease activity in the larval stage of the parasitoid wasp, Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae); effects of protease inhibitors.

    Down, R E; Ford, L; Mosson, H J; Fitches, E; Gatehouse, J A; Gatehouse, A M

    1999-08-01

    Hymenopteran, parasitoid wasps have good potential for use in integrated pest management (IPM); for example, the gregarious ectoparasitoid, Eulophus pennicornis, has been suggested as a biological control agent for larvae of the tomato moth (Lacanobia oleracea L.). However, the processes by which such parasitic larvae are able to utilize the nutritional resource provided by the host have been little studied. Protease activity was present in E. pennicornis larvae, and characterization of the enzymes responsible for proteolysis was performed using a range of synthetic substrates and specific inhibitors. Serine protease enzymes was both trypsin- and chymotrypsin-like activities were present. A range of plant-derived serine protease inhibitors was tested for activity against these enzymes. Certain inhibitors, notably soybean Kunitz inhibitor (SKTI), inhibited enzyme activity by > 80% at pests are to form a component of IPM systems, possible adverse effects, whether direct or indirect, of transgene expression on parasitoids like E. pennicornis should be considered. PMID:10466123

  1. Co-lethality studied as an asset against viral drug escape: the HIV protease case

    Ollivier Emmanuelle

    2010-06-01

    Full Text Available Abstract Background Co-lethality, or synthetic lethality is the documented genetic situation where two, separately non-lethal mutations, become lethal when combined in one genome. Each mutation is called a "synthetic lethal" (SL or a co-lethal. Like invariant positions, SL sets (SL linked couples are choice targets for drug design against fast-escaping RNA viruses: mutational viral escape by loss of affinity to the drug may induce (synthetic lethality. Results From an amino acid sequence alignment of the HIV protease, we detected the potential SL couples, potential SL sets, and invariant positions. From the 3D structure of the same protein we focused on the ones that were close to each other and accessible on the protein surface, to possibly bind putative drugs. We aligned 24,155 HIV protease amino acid sequences and identified 290 potential SL couples and 25 invariant positions. After applying the distance and accessibility filter, three candidate drug design targets of respectively 7 (under the flap, 4 (in the cantilever and 5 (in the fulcrum amino acid positions were found. Conclusions These three replication-critical targets, located outside of the active site, are key to our anti-escape strategy. Indeed, biological evidence shows that 2/3 of those target positions perform essential biological functions. Their mutational variations to escape antiviral medication could be lethal, thus limiting the apparition of drug-resistant strains. Reviewers This article was reviewed by Arcady Mushegian, Shamil Sunyaev and Claus Wilke.

  2. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

    Kugadas, Abirami; Lamont, Elise A; Bannantine, John P; Shoyama, Fernanda M; Brenner, Evan; Janagama, Harish K; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  3. Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases

    Malhotra Pawan; Chauhan Virander S; Sunil Sujatha

    2008-01-01

    Abstract Background Plasmodium falciparum cysteine proteases (falcipains) play indispensable roles in parasite infection and development, especially in the process of host erythrocyte rupture/invasion and hemoglobin degradation. No detailed molecular analysis of transcriptional regulation of parasite proteases especially cysteine proteases has yet been reported. In this study, using a combination of transient transfection assays and electrophoretic mobility shift assays (EMSA), we demonstrate...

  4. Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

    Ladislau C. Kovari

    2012-05-01

    Full Text Available Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates.

  5. Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease

    Johnson, R. Jeremy

    2014-01-01

    HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure-based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a…

  6. A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

    Halls, C.E.; Rogers, S. W.; Ouffattole, M.;

    2006-01-01

    proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts, it...

  7. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases. PMID:26178876

  8. PENGEMPUKAN DAGING DENGAN ENZIM PROTEASE TANAMAN BIDURI (Calotropis gigantea) [Meat Tenderization using Protease of Biduri Plant (Calotropis gigantea)

    Erni Sofia Murtini1); Qomarudin2)

    2003-01-01

    Tenderness is the main attribute quality of meat, which influences consumer acceptability. Protease enzyme (like papain, bromelin and ficin) are known to be used for improving tenderness of meat trough degradation of the protein. Biduri plant (Calotropis gigantea) contains protease enzyme in its latex or the young tissue (0-20 cm plant tip). After isolation of crude enzyme using ammonium sulphate, the enzyme was the applied to tenderise meat at concentrations 0 ; 0,25; 0,5; 0,75 and 1,0%. The...

  9. Serine protease from midgut of Bombus terrestris males

    Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

    2013-01-01

    Roč. 82, č. 3 (2013), s. 117-128. ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

  10. Tomato ringspot nepovirus protease: characterization and cleavage site specificity

    Hans, F.; Sanfacon, H.

    1995-01-01

    We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have dem

  11. Efficiency of hydrolysis of whey protein serine proteases

    Г.П. Петюх; Романова, Н.А.; К.М. Гаркава

    2009-01-01

     Research of relative enzymatic whey protein isolate (WPI) hydrolysis efficiency was conducted. Seven enzymes in three concentrations by pH 7.0 t=50°C were used. The most efficient showed to be Protease R in concentration 0,0002 g/ml.

  12. Manipulating the autolytic pathway of a Bacillus protease

    VandenBurg, B; Eijsink, VGH; Vriend, G; Veltman, OR; Venema, G; HopsuHavu, VK; Jarvinen, M; Kirschke, H

    1997-01-01

    Autolytic degradation of Bacillus subtilis thermolysin-like proteinase (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously, we reported five autolysis sites in B. subtilis neutral protease (Van den Burg et al., 1990, Biochem. J. 272:93-97). I

  13. Protease determination using an optimized alcohol enzyme electrode.

    Bardeletti, G; Carillon, C

    1993-12-01

    A new method for the determination of protease activities is described. In this large family, trypsin is used as a protease model that cleaves the ethyl or methyl ester of artificial substrates producing ethanol or methanol. Alcohol is detected using an alcohol oxidase enzyme electrode. The H2O2 production that occurs is measured amperometrically. At 30 degrees C, in a 0.1M phosphate buffer, pH 7.5, the enzyme electrode response for ethanol was calibrated at 3.10(-6)-3.10(-3)M and for methanol from 3.10(-7) to 4.10(-4)M in the cell measurement. Trypsin levels as determined by the proposed method and by a conventional spectrophotometric method are in good agreement when using the same measurement conditions. A detection limit of 10 U.L-1 and a linear calibration curve of 10-100,000 U.L-1 in the sample were obtained. Measuring time for the required trypsin solution concentration was from 4 min (for the most dilute samples) to 1 min (for the most concentrate samples). In a typical experiment, protease measurements did not inactivate the alcohol oxidase on the probe, nor did a more classical use for alcohol detection. The procedure developed could permit any protease estimation on the condition that they hydrolyze ester bonds from synthetic substrate. PMID:8109959

  14. Intervention with Serine Protease Activity with Small Peptides

    Xu, Peng

    2015-01-01

    , plasma kallikrein, which contributes to the pathogenesis in hereditary angioedema. According to the X-ray crystal structure analysis, we proposed a principle for designing inhibitors of other serine proteases from mupain-1. In order to be able to evaluate the inhibitory activities of our peptides in vivo...

  15. Breakdown of the innate immune system by bacterial proteases

    Laarman, A.J.

    2011-01-01

    Bacteria have developed many strategies to circumvent our immune system to survive and colonize human tissues. One of these strategies is by secreting proteases that specifically target the innate immune system. Aureolysin is a metalloprotease from Staphylococcus aureus which target the main compone

  16. Ionic liquids and proteases: A clean alliance for semisynthesis

    Wehofsky, N.; Wespe, Ch.; Čeřovský, Václav; Pech, A.; Hoess, E.; Rudolph, R.; Bordusa, F.

    2008-01-01

    Roč. 9, č. 9 (2008), s. 1493-1499. ISSN 1439-4227 Grant ostatní: DFG(DE) SPP1191; DFG(DE) SFB610 Institutional research plan: CEZ:AV0Z40550506 Keywords : chemoenzymatic synthesis * ionic liquids * peptides * proteases * substrate mimetics Subject RIV: CC - Organic Chemistry Impact factor: 3.322, year: 2008

  17. Retroviral proteases and their roles in virion maturation

    Konvalinka, Jan; Kräusslich, H. G.; Müller, B.

    2015-01-01

    Roč. 479, SI (2015), s. 403-417. ISSN 0042-6822 R&D Projects: GA ČR GBP208/12/G016; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : retrovirus * aspartic protease * maturation * human immunodeficiency virus * Gag Subject RIV: CE - Biochemistry Impact factor: 3.321, year: 2014

  18. THE ROLE OF CYSTEINE PROTEASE IN ALZHEIMER DISEASE

    Hasanbasic, Samra; Jahic, Alma; Karahmet, Emina; Sejranic, Asja; Prnjavorac, Besim

    2016-01-01

    Introduction: Cysteine protease are biological catalysts which play a pivotal role in numerous biological reactions in organism. Much of the literature is inscribed to their biochemical significance, distribution and mechanism of action. Many diseases, e.g. Alzheimer’s disease, develop due to enzyme balance disruption. Understanding of cysteine protease’s disbalance is therefor a key to unravel the new possibilities of treatment. Cysteine protease are one of the most important enzymes for protein disruption during programmed cell death. Whether protein disruption is part of cell deaths is not enough clear in any cases. Thereafter, any tissue disruption, including proteolysis, generate more or less inflammation appearance. Review: This review briefly summarizes the current knowledge about pathological mechanism’s that results in AD, with significant reference to the role of cysteine protease in it. Based on the summary, new pharmacological approach and development of novel potent drugs with selective toxicity targeting cysteine protease will be a major challenge in years to come.

  19. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621. ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity -based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 3.088, year: 2014

  20. In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

    Jeong Hee Kim

    Full Text Available This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s. The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

  1. Evidence-Performance-Gap bei Typ-2-Diabetikern in Schweizer Hausarztpraxen: Sind Patienten mit einem HbA1c über 7% tatsächlich schlecht eingestellt?

    Djalali, Sima; Frei, Anja

    2013-01-01

    Diabetes-Typ-2 Patienten mit einem höheren HbA1c sind älter, schwerer, leiden schon länger unter der Krankheit und eher unter Komorbiditäten als Patienten mit einem tieferen HbA1c. Oder doch nicht? Eine Studie in Schweizer Hausarztpraxen brachte Überraschendes zu Tage.

  2. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  3. Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt–Jakob disease: a case report

    Rodríguez-Martínez Ana B

    2012-10-01

    Full Text Available Abstract Introduction The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation A 74-year-old Caucasian woman showed a sporadic Creutzfeldt–Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient’s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt–Jakob disease. This highlights the

  4. A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

    Marta Gogliettino

    2014-02-01

    Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

  5. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    Tripathi Lokesh P

    2008-11-01

    Full Text Available Abstract Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc. were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes

  6. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  7. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  8. PEMURNIAN PROTEASE DARI BUAH DAN DAUN MENGKUDU (Morinda citrifolia L.) [Purification of Proteases from Fruits and Leaves of Noni (Morinda citrifolia L.)

    Dwi Ishartani1,2)*; Elfi2); Nuri Andarwulan1,3); Dahrul Syah1,2)*

    2011-01-01

    Proteases have been widely used in cancer treatment, wounds healing, overcoming digestion disorder and other modern pharmaceutical applications. Proteases may be present in fruits and leaves of noni (Morinda citrifolia L.) since the plant has been used traditionally in wound healing. This research aimed to purify proteases from noni’s fruits and leaves at two maturity stages, i.e. fruits with green-yellow (TK2) and white-yellow (TK4) skin colour, also leaves from shoot and base. Proteases wer...

  9. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  10. Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

    Zahra Ghobadi Nejad

    2014-01-01

    Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

  11. IgA Protease Activity in Haemophilus parasuis in the Absence of a Recognizable IgA Protease Gene

    Background. Haemophilus parasuis, the bacterium responsible for Glasser’s disease, is a pathogen of significant concern in modern high-health swine production systems. Little is known regarding the molecular mechanisms of H. parasuis infection. In some Pasteurellaceae species, IgA proteases aid in d...

  12. Enhanced long-term and impaired short-term spatial memory in GluA1 AMPA receptor subunit knockout mice: Evidence for a dual-process memory model

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of GluA1-dependent synaptic plasticity for short-term memory of recently visited places, but not for the ability to form long-term associations betwee...

  13. Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease fromArenicola cristata

    ZHAO Chunling; JU Jiyu

    2015-01-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplifi-cation of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encod-ing a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (<40%) to other serine proteases. The gene encoding the active form ofA. cristata serine protease was cloned and expressed inE. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result sug-gested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clotin vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene inA. cristata.

  14. In silico prediction of mutant HIV-1 proteases cleaving a target sequence

    Jensen, Jan H; Winther, Jakob R; De Vico, Luca

    2014-01-01

    HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636 -- 1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidate...

  15. Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology.

    Homaei, Ahmad; Lavajoo, Fatemeh; Sariri, Reyhaneh

    2016-07-01

    Marine environment consists of the largest sources diversified genetic pool of material with an enormous potential for a wide variety of enzymes including proteases. A protease hydrolyzes the peptide bond and most of proteases possess many industrial applications. Marine proteases differ considerably from those found in internal or external organs of invertebrates and vertebrates. In common with all enzymes, external factors such as temperature, pH and type of media are important for the activity, catalytic efficiency, stability and proper functioning of proteases. In this review valuable characteristics of proteases in marine organisms and their applications are gathered from a wide literature survey. Considering their biochemical significance and their increasing importance in biotechnology, a thorough understanding of marine proteases functioning could be of prime importance. PMID:27086293

  16. Peripheral lipodystrophy measure by DEXA in patients receiving HIV protease inhibitors

    Full text: Protease inhibitors in combination with HIV reverse transcriptase inhibitors are now recommended as standard anti-retroviral therapy. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance, which correlates closely with abdominal obesity, has been associated with HIV protease inhibitors. 62 HIV-infected patients, 43 receiving protease inhibitors and 19 protease inhibitor-naive, had dual energy X-ray absorptiometry (DEXA) measurements using the Lunar DPXL performed of the total body to assess body fat distribution. Each group had a comparable mean age and body mass index. Patients receiving protease inhibitors had significantly lower mean fat mass overall and in each region except for the central abdomen when compared to the protease inhibitor-naive group. This study demonstrates that DEXA total body measurement using the Lunar DPXL may be a useful tool in detecting regionalized fat loss in HIV positive patients receiving protease inhibitors and has potential for assessing its therapy

  17. Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

    Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

  18. Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

    Senthilvel, Padmanaban; Lavanya, Pandian; Kumar, Kalavathi Murugan; Swetha, Rayapadi; Anitha, Parimelzaghan; Bag, Susmita; Sarveswari, Sundaramoorthy; Vijayakumar, Vijayaparthasarathi; Ramaiah, Sudha; Anbarasu, Anand

    2013-01-01

    Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-...

  19. The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

    Yu-Yin Li

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

  20. Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

    Little Tom J; Welch John J; Obbard Darren J

    2009-01-01

    Abstract Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously be...

  1. Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora

    Tunlid, Anders; Jansson, Sven

    1991-01-01

    The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substra...

  2. Pathogenic capacity of proteases from Serratia marcescens and Pseudomonas aeruginosa and their suppression by chicken egg white ovomacroglobulin.

    Molla, A; Matsumura, Y; Yamamoto, T.; Okamura, R.; H. Maeda

    1987-01-01

    The pathogenicities of three proteases from Serratia marcescens, two proteases from Pseudomonas aeruginosa, and one thermolysin from Bacillus stearothermophilus were examined. All proteases tested caused acute liquefactive necrosis of the cornea and descemetocele formation in guinea pig eyes after intrastromal injection, with the exception of the 60-kilodalton protease from S. marcescens, which produced only an opaque lesion. When injected into guinea pig skin, the protease also enhanced vasc...

  3. Mutation in type II procollagen (COL2A1) that substitutes aspartate for glycine alpha 1-67 and that causes cataracts and retinal detachment: evidence for molecular heterogeneity in the Wagner syndrome and the Stickler syndrome (arthro-ophthalmopathy)

    Körkkö, J; Ritvaniemi, P; Haataja, L; Kääriäinen, H; Kivirikko, K I; Prockop, D J; Ala-Kokko, L

    1993-01-01

    A search for mutations in the gene for type II procollagen (COL2A1) was carried out in affected members of a family with early-onset cataracts, lattice degeneration of the retina, and retinal detachment. They had no symptoms suggestive of involvement of nonocular tissues, as is typically found in the Stickler syndrome. The COL2A1 gene was amplified with PCR, and the products were analyzed by denaturing gradient gel electrophoresis. The results suggested a mutation in one allele for exon 10. S...

  4. Occurrence of aspartyl proteases in brine after herring marinating.

    Szymczak, Mariusz; Lepczyński, Adam

    2016-03-01

    Herrings are marinated in a brine consisting of salt and acetic acid. During marinating, various nitrogen fractions diffuse from fish flesh to the brine, causing significant nutritional quality losses of the raw material. In this study, it has been demonstrated for the first time that proteases diffuse from the fish to the marinating brine. Using ammonium sulphate precipitation and affinity chromatography on pepstatin-A agarose bed the aspartyl proteases were purified and concentrated over 2600-fold from a marinating brine. Pepstatin-A completely inhibited the activity of the purified preparation. The preparation was active against fluorogenic substrates specific for cathepsin D and E and inactive against substrates specific for cysteine cathepsins. Depending on incubation time, the preparation showed pH-optimum at 2.0 or 4.5. The 2D SDS-PAGE separation demonstrated the presence of a few proteins with molecular weights and pI values typical of cathepsin D, E and pepsin. PMID:26471581

  5. Stability and selectivity of alkaline proteases in hydrophilic solvents

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    substitution. Some of the most abundant hexoses were all substituted at the primary hydroxyl group at the C-6 position in processes catalysed by different alkaline proteases [3,4,5]. However by adding DMSO to the reaction medium the regio-selectivity in a Streptomyces sp protease catalysed reaction was shifted...... was 10 minutes. The activity was effected by the solvation of the enzyme in both DMSO and DMF [11]. Literature   [1]           H. Ogino, H. Ishikawa, J. Biosci. Bioeng. 2001, 91, 109. [2]           K. Watanabe, S. Ueji, Biotechnol. Lett. 2000, 22, 599. {3]           M. Kitagawa, H. Fan, T. Raku, S...

  6. The role of lysosomal cysteine proteases in crustacean immune response

    FL Garcia-Carreño

    2014-04-01

    Full Text Available Over the long course of evolution and under the selective pressure exerted by pathogens and parasites, animals have selectively fixed a number of defense mechanisms against the constant attack of intruders. The immune response represents a key component to optimize the biological fitness of individuals. Two decades ago, prevention and control of diseases in crustacean aquaculture systems were considered priorities in most shrimp-producing countries, but knowledge was scarce and various pathogens have severely affected aquaculture development around the world. Scientific contributions have improved our understanding of the crustacean immune response. Several studies confirm the central role played by proteases in the immune response of animals, and the cooperative interaction of these enzymes in a wide variety of organisms is well known. This review summarizes the current information regarding the role of cysteine proteases in the immune system of Crustacea and points to aspects that are needed to provide a better integration of our knowledge.

  7. PLANT PROTEASE INHIBITORS: STRATEGY FOR PEST CONTROL IN CROPS

    R.S.DHANDE1 N.J.CHIKHALE2

    2014-11-01

    Full Text Available Proteinase inhibitors (PIs are naturally occurring proteins in living organisms and are able to inhibit & control the activity of proteases. PIs are a diverse group of proteins that share a common biochemical activity. The role of plant proteinase inhibitors was investigated by Mickel and Standish in 1947 when they observed the insects larvae were unable to develop normally on soybean products. Subsequently, the soybean trypsin inhibitors were found to be lethal to the flour beetle larvae, Tribolium confusum (Lipke et. al., 1954. Now there are diverse examples of protease inhibitors active against many insect species both in vitro (Pannetier et. al., 1997; Koiwa et. al., 1998 and in vivo (Urwin et. al., 1997; Vain et. al., 1998 bioassays.

  8. Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses

    Gottwein, Judith M; Scheel, Troels K H; Jensen, Tanja B; Ghanem, Lubna; Bukh, Jens

    2011-01-01

    The hepatitis C virus (HCV) genotype influences efficacy of interferon (IFN)-based therapy. HCV protease inhibitors are being licensed for treatment of genotype 1 infection. Because there are limited or no data on efficacy against HCV genotypes 2-7, we aimed at developing recombinant infectious...... cell culture systems expressing genotype-specific nonstructural (NS) protein 3 protease (NS3P)....

  9. Cysteine proteases: Modes of activation and future prospects as pharmacological targets

    Sonia eVerma

    2016-04-01

    Full Text Available Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria and parasite to the higher organisms (mammals. Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases and metallo-proteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a pro-domain (regulatory and a mature domain (catalytic. The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.

  10. Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii W6b

    LI Jing; PENG Ying; WANG Xianghong; CHI Zhenming

    2010-01-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease.The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 ℃.The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts.The optimal medium of the acid protease production was seawater containing 1.0% glucose,1.5% casein,and 0.5% yeast extract,and the optimal cultivation conditions of the acid protease production were pH 4.0,a temperature of 25 ℃ and a shaking speed of 140 rmin-1.Under the optimal conditions,72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level.The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources.Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability.The acid protease produced by M.reukaufii W6b may have highly potential applications in cheese,food and fermentation industries.

  11. Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of Acanthamoeba

    Khan, Naveed A.; Jarroll, Edward L.; Panjwani, Noorjahan; Cao, Zhiyi; Paget, Timothy A.

    2000-01-01

    Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5. PMID:10921939

  12. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  13. Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

    Lai Meng-Jiun

    2010-08-01

    Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

  14. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  15. Characterization of a membrane-associated serine protease in Escherichia coli

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [3H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

  16. Isolation and characterization of protease from Bacillus subtilis 1012M15

    ELFI SUSANTI

    2003-01-01

    Full Text Available A local strain of Bacillus sp. BAC4, is known to produce penicillin G acylase (PGA enzyme with relatively high activity. This strain secretes the PGA into the culture medium. However, it has been reported that PGA activity fall and rise during culture, and the activity plummets during storege at –200C, which probably due to usage protease activity of Bacillus sp. BAC4. To study the possible use of Bacillus subtilis 1012M15 as a host cell for cloning the pga gene from Bacillus sp. BAC4, the protease activity of Bacillus subtilis 1012M15 were studied. Protease activity was determined by Horikoshi method. In this experiment, maximum protease activity in Bacillus subtilis 1012M15 culture was obsereved after 8 hours. At this optimum condition, protease activity of Bacillus sp. BAC4 is five time higher than that of Bacillus subtilis 1012M15. This situation promised the possible usage of Bacillus subtilis 1012M15 as a host cell for pga expression. For protease characterization, the bacterial culture had been separated from the cell debris by centrifugation. The filtrate was concentrated by freeze drying, fractionated by ammonium sulphate, dialyzed in selovan tube, and then fractionated by ion exchance chromatography employing DEAE-cellulose. The five peaks resulted indicated the presence of five protease. Based on inhibitor and activator influence analysis, it could be concluded that proteases from Bacillus subtilis 1012M15 contained of serin protease as well as metalloprotease and serin protease mixture.

  17. An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

    Piero Giansanti; Thin Thin Aye; Henk van den Toorn; Mao Peng; Bas van Breukelen; Albert J.R. Heck

    2015-01-01

    Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti4+-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substa...

  18. Acute Pancreatitis. Studies on smoking and protease activation.

    Lindkvist, Björn

    2005-01-01

    Background and aims: Activation of pancreatic proteases is considered to be a crucial event in the early phase of acute pancreatitis but the cause of this activation is not known. Most cases of acute pancreatitis can be attributed to either gallstone disease or alcohol abuse. However, little is known about other risk factors. The aim of this thesis is to investigate the mechanisms involved in the initiation of acute pancreatitis, trends in the incidence, and risk factors for the disease. The ...

  19. Schistosome serine protease inhibitors: parasite defense or homeostasis?

    Landys A. Lopez Quezada

    2011-06-01

    Full Text Available Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL. Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.Serpinas são uma família de inibidores macromoleculares estruturalmente conservados encontrados em inúmeros sistemas biológicos. O término e a anotação dos genomas de Schistosoma mansoni e de Schistosoma japonicum permitiram a identificação por análise filogenética de dois principais clados de serpinas. S. mansoni mostra uma multiplicidade maior de genes de serpinas, talvez refletindo uma adaptação à infecção de um hospedeiro humano. Alvos putativos das serpinas de esquistossomos podem ser preditos a partir da sequência do "loop" do centro reativo. Serpinas de esquistossomos podem ter importantes papeis tanto na regulação pós-traducional de proteases derivadas do esquistossoma, quanto nos mecanismos de defesa contra a ação de proteases do hospedeiro.

  20. Operating Conditions Effects Onenzyme Activity: Case Enzyme Protease

    Adel Oueslati,; Mounirhaouala

    2014-01-01

    The Proteases an enzyme added to detergents to degrade the protein spots origin.Their action is manifested through its activity the middle of washing clothes. This activity depends on the operating conditions. In this article, the effects of temperature and pH of the reaction and the substrate concentration and time of washing medium on the enzyme activity were studied. There action mechanism has been shown. The activity measurements were made by absorption spectrometry

  1. Venomous protease of aphid soldier for colony defense

    Kutsukake, Mayako; Shibao, Harunobu; Nikoh, Naruo; Morioka, Mizue; Tamura, Tomohiro; Hoshino, Tamotsu; Ohgiya, Satoru; Fukatsu, Takema

    2004-01-01

    In social aphids, morphological, behavioral, and physiological differences between soldiers and normal insects are attributed to differences in gene expression between them, because they are clonal offspring parthenogenetically produced by the same mothers. By using cDNA subtraction, we identified a soldier-specific cysteine protease of the family cathepsin B in a social aphid, Tuberaphis styraci, with a second-instar soldier caste. The cathepsin B gene was specifically expressed in soldiers ...

  2. HIV protease inhibitors in gut barrier dysfunction and liver injury

    Wu, Xudong; Li, Yunzhou; Peng, Kesong; Zhou, Huiping

    2014-01-01

    The development of HIV protease inhibitors (HIV PIs) has been one of the most significant advances of the past two decades in controlling HIV infection. HIV PIs have been used successfully in highly active anti-retroviral therapy (HAART) for HIV infection, which is currently the most effective treatment available. Incorporation of HIV PIs in HAART causes profound and sustained suppression of viral replication, significantly reduces the morbidity and mortality of HIV infection, and prolongs th...

  3. The Early Years of Retroviral Protease Crystal Structures

    Miller, Maria

    2010-01-01

    Soon after its discovery, the attempts to develop anti-AIDS therapeutics focused on the retroviral protease (PR) — an enzyme used by lentiviruses to process the precursor polypeptide into mature viral proteins. An urgent need for the three-dimensional structure of PR to guide rational drug design prompted efforts to produce milligram quantities of this enzyme. However, only minute amounts of PR were present in the HIV-1 and HIV-2 viruses, and initial attempts to express this protein in bacter...

  4. Serine proteases mediate inflammatory pain in acute pancreatitis

    Ceppa, Eugene P; Lyo, Victoria; Grady, Eileen F.; Knecht, Wolfgang; Grahn, Sarah; Peterson, Anders; Nigel W. Bunnett; Kirkwood, Kimberly S.; Cattaruzza, Fiore

    2011-01-01

    Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR2), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR2 and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Inje...

  5. Purification and characterization of an extracellular protease of Legionella pneumophila.

    Dreyfus, L A; Iglewski, B H

    1986-01-01

    An extracellular proteolytic enzyme of Legionella pneumophila was purified by sequential batch separation with DEAE-cellulose, hydrophobic interaction chromatography with octyl-Sepharose, and ion-exchange chromatography with DEAE-Bio-Gel A (Bio-Rad Laboratories, Richmond, Calif.). The resulting protease preparation was determined to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Although free of contaminating proteins, the purified ...

  6. Preparation of lignin derivatives and their application as protease adsorbents

    Xian-Su Cheng; Yin Lin; Run Fang

    2009-01-01

    Synthesis of two lignin derivatives, lignophenol and lignin-aminophenol, were presented in this article. The chemical structure and the func-tional groups of lignin derivatives were charac-terized through FT-IR analysis. The immobiliza-tion of three proteases (papain, trypsin and pepsin) on lignin and lignin derivatives was carried out using adsorption technique. The influence of contact time and pH on the enzyme adsorption by different adsorbents was inves-tigated. Furthermore, enzyme activi...

  7. Protease activation in glycerol-based deep eutectic solvents

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2011-01-01

    Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually exa...

  8. Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3

    de Rozières Sohela

    2004-11-01

    Full Text Available Abstract Background The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus, and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. Results Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05 higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05. Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease. Conclusions The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of

  9. Studies on the disulfide region of α1-protease inhibitor

    The single disulfide bond of purified human α1-protease inhibitor was reduced with dithiothreitol in the absence of denaturant and the resultant sulfhydryl groups were alkylated with iodoacetamide-1-C14. The product was found to be fully functional as an inhibitor of trypsin and elastase in esterolytic and proteolytic assays. The modified protein was also found to be nearly identical to native α1-protease inhibitor when analyzed by immunological, electrophoretic, and spectral methods. The performic acid oxidized inhibitor, on the other hand, was devoid of any enzyme inhibitory activity. Analysis of the derivatized protein by amino acid analysis and by radioactive counting revealed only a single cysteine-containing peptide. The alkylated inhibitor was digested with cyanogen bromide and then trypsin, and subjected to two-dimensional peptide mapping. A single cysteine-containing peptide was recovered and shown to have the sequence Phe-Asn-Ile-Gln-His-Cys-Lys. A variety of experiments involving gel filtration or dialysis of reduced or oxidized α1-protease inhibitor indicate that this Cyspeptide is covalently bound to either free cysteine or to glutathione via a disulfide bridge. (author)

  10. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

    Nilesh P. Nirmal

    2014-01-01

    Full Text Available A fungal strain (Conidiobolus brefeldianus MTCC 5184 isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50% and sorbitol (50% at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory.

  11. Detection of extracellular proteases from microorganisms on agar plates

    Alane Beatriz Vermelho

    1996-12-01

    Full Text Available We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

  12. Effect of protease supplementation on production performance of laying hens

    Javer Alves Vieira Filho

    2015-02-01

    Full Text Available The experiment was conducted with the objective of evaluating the effect of protease enzyme supplementation on performance parameters and quality shell of laying hens. We used 240 Isa Brown commercial laying hens with 44 weeks of age. A completely randomized split-plot design (5 periods of 21 days with four treatments and six replications (10 hens per replication was used. The experimental diets were formulated according to the requirements of the breed. The following parameters were evaluated: egg production, feed intake, feed conversion, average egg weight, egg loss, specific gravity, percentage and shell thickness. After collection, the data were analyzed with SISVAR Statistical Package, and the means compared by SNK test at 5% probability. It was concluded that supplementation of diets low in nutrients with 500 g ton-1 of protease (100 U g-1, provides egg production and feed conversion rates similar to those obtained in laying hens fed diet with the nutritional level recommended for the breed. However, protease supplementation did not show effect on egg shell quality.

  13. New roles for perforins and proteases in apicomplexan egress.

    Roiko, Marijo S; Carruthers, Vern B

    2009-10-01

    Egress is a pivotal step in the life cycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late-stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called serine-repeat antigens that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans. PMID:19614666

  14. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

    Surasak Jittavisutthikul

    2015-04-01

    Full Text Available There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN. Human hepatic (Huh7 cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β, indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.

  15. Protease activated receptors (PARS) mediation in gyroxin biological activity

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH2, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  16. Exoerythrocytic Plasmodium parasites secrete a cysteine protease inhibitor involved in sporozoite invasion and capable of blocking cell death of host hepatocytes.

    Annika Rennenberg

    2010-03-01

    Full Text Available Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases. We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.

  17. The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

    Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga; Pachl, Petr; Pichová, Iva; Řezáčová, Pavlína

    2012-01-01

    Roč. 27, č. 1 (2012), s. 160-165. ISSN 1475-6366 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : secreted aspartic protease * virulence factor * X-ray structure * candidiasis Subject RIV: CE - Biochemistry Impact factor: 1.495, year: 2012

  18. Thermodynamic and structural analysis of HIV protease resistance to darunavir - analysis of heavily mutated patient- derived HIV-1 proteases

    Kožíšek, Milan; Lepšík, Martin; Grantz Šašková, Klára; Brynda, Jiří; Konvalinka, Jan; Řezáčová, Pavlína

    2014-01-01

    Roč. 281, č. 7 (2014), s. 1834-1847. ISSN 1742-464X R&D Projects: GA ČR GAP207/11/1798 Grant ostatní: OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : enthropic contribution * HIV protease inhibitors * isothermal titration calorimetry * resistance mutation * X-ray crystallography Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

  19. GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions

    Grantz Šašková, Klára; Kožíšek, Milan; Stray, K.; Jong de, D.; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; Nijhuis, M.; Cihlář, T.; Konvalinka, Jan

    2014-01-01

    Roč. 88, č. 6 (2014), s. 3586-3590. ISSN 0022-538X R&D Projects: GA ČR GAP207/11/1798 Grant ostatní: OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : virus type-1 protease * antiviral activity * drug resistance Subject RIV: EE - Microbiology, Virology Impact factor: 4.439, year: 2014

  20. Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

    Luciana Bonome Zeminian

    2013-02-01

    Full Text Available The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3 have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

  1. The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition*

    Azarkan, Mohamed; Martinez-Rodriguez, Sergio; Buts, Lieven; Baeyens-Volant, Danielle; Garcia-Pino, Abel

    2011-01-01

    Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition. PMID:22027836

  2. Proteases and antiproteases in chronic neutrophilic lung disease - relevance to drug discovery.

    Greene, Catherine M

    2009-10-01

    Chronic inflammatory lung diseases such as cystic fibrosis and emphysema are characterized by higher-than-normal levels of pulmonary proteases. While these enzymes play important roles such as bacterial killing, their dysregulated expression or activity can adversely impact on the inflammatory process. The existence of efficient endogenous control mechanisms that can dampen or halt this overexuberant protease activity in vivo is essential for the effective resolution of inflammatory lung disease. The function of pulmonary antiproteases is to fulfil this role. Interestingly, in addition to their antiprotease activity, protease inhibitors in the lung also often possess other intrinsic properties that contribute to microbial killing or termination of the inflammatory process. This review will outline important features of chronic inflammation that are regulated by pulmonary proteases and will describe the various mechanisms by which antiproteases attempt to counterbalance exaggerated protease-mediated inflammatory events. These proteases, antiproteases and their modifiers represent interesting targets for therapeutic intervention.

  3. Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes

    Glushakova, Svetlana; Mazar, Julia; Hohmann-Marriott, Martin F; Hama, Erinn; Zimmerberg, Joshua

    2008-01-01

    By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells, and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of...

  4. Novel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine Proteases

    Verhelst, Steven H. L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo, Matthew

    2006-01-01

    Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing O-acylhydroxamates that form stable covalent adducts with target proteases. This allows them to be used as ABPs for papain family cysteine proteases. A second class of related analogues containing ...

  5. α-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB

    Steert, Koen; Berg, Maya; Mottram, Jeremy C.; Westrop, Gareth D.; Coombs, Graham H; Cos, Paul; Maes, Louis; Joossens, Jurgen; Van der Veken, Pieter; Haemers, Achiel; Augustyns, Koen

    2010-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of α-ketoheterocycles 1-15 has been developed as reversible inhibitors of a r...

  6. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    Marton Siklos; Manel BenAissa; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families...

  7. Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of leishmania mexicana cysteine protease CPB

    Schroder, J.; S. Noack; Marhofer, R.J.; Mottram, J. C.; Coombs, G.H.; Selzer, P.M.

    2013-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput sc...

  8. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and...

  9. Falstatin, a Cysteine Protease Inhibitor of Plasmodium falciparum, Facilitates Erythrocyte Invasion

    Pandey, Kailash C.; Singh, Naresh; Arastu-Kapur, Shirin; Bogyo, Matthew; Rosenthal, Philip J.

    2006-01-01

    Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi c...

  10. OPTIMIZATION OF EXTRACELLULAR ACID PROTEASE PRODUCTION FROM ASPERGILLUS NIGER BY FACTORIAL DESIGN

    Vishalkirti Vijay Kalaskar; Narayanan Kasinathan; Volety Mallikarjuna Subrahmanyam; Josyula Venkata Rao

    2014-01-01

    The cultural conditions for acid protease production by Aspergillus niger was optimised using factorial design experiments and one factor-at-a time approach. In the production medium casein served as substrate and protease activity was measured in terms of tyrosine yield. The yield was further improved through UV mutation. Tyrosine yield amounted to 29.22 mg / g on casein substrate. Protease from this microbial strain was mesophilic. The enzyme was stable over a wide temperature range (30 to ...

  11. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    Jong W Yu

    Full Text Available CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  12. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    Huiyun Zhang; Xiaoning Zeng; Shaoheng He

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process...

  13. The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

    Vergis, James M.; Michael C. Wiener

    2011-01-01

    Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase...

  14. Purification and characterization of a serine protease (CESP) from mature coconut endosperm

    Mandal Chhabinath; Roy Samir; Usha Rajamma; Panicker Leelamma M

    2009-01-01

    Abstract Background In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm. Findings We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate...

  15. The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme

    Lindner, Holger A.; Fotouhi-Ardakani, Nasser; Lytvyn, Viktoria; Lachance, Paule; Sulea, Traian; Ménard, Robert

    2005-01-01

    The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. ...

  16. Evaluation of antifungal activity of protease inhibitors from potato (Solanum tuberosum L.)

    REISEROVÁ, Jana

    2014-01-01

    This diploma thesis is concerned on protease inhibitors isolated from potato (Solanum tuberosum L.) tubers and evaluation of their antifungal properties. Theoretical part of the thesis deals with protease inhibitors which have an antifungal effect. Tubers of potato cultivars Adéla, Ornella, Eurostarch - were used for protease inhibitors isolation. Antifungal activity of isolated protein fractions were evaluated versus fungi from genus Rhizoctonia and Fusarium that are important pathogens in a...

  17. Impact of Host Proteases on Reovirus Infection in the Respiratory Tract

    Nygaard, Rachel M.; Golden, Joseph W.; Schiff, Leslie A.

    2012-01-01

    Virion uncoating is an essential early event in reovirus infection. In natural enteric infections, rapid proteolytic uncoating of virions is mediated by pancreatic serine proteases. The proteases that promote reovirus disassembly and cell entry in the respiratory tract remain unknown. In this report, we show that endogenous respiratory and inflammatory proteases can promote reovirus infection in vitro and that preexisting inflammation augments in vivo infection in the murine respiratory tract.

  18. Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties.

    Jackman, D M; Bartlett, F M; Patel, T R

    1983-01-01

    A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic det...

  19. Use of MALDI-TOF Mass Spectrometry for Specificity Studies of Biomedically Important Proteases

    Siigur, Jüri; Trummal, Katrin; TÕnismägi, Külli; Samel, Mari; Siigur, Ene; Vija, Heikki; Tammiste, Indrek; Subbi, Juhan

    2002-01-01

    Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viper Vipera lebetina, Common viper Vipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activa...

  20. Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation.

    Wang, P L; Shirasu, S; Shinohara, M; Daito, M; Oido, M; Kowashi, Y; Ohura, K

    1999-04-01

    Proteases produced by Porphyromonas gingivalis are believed to contribute to the pathogenesis of periodontal diseases. Here the cytotoxic effects of a purified preparation of a P. gingivalis protease with trypsin-like specificity were tested on human gingival fibroblasts in vitro. The active protease induced apoptotic cell death in the fibroblasts, as indicated by DNA fragmentation and the expression of 7A6 antigen. Thus, the production of proteases by periodontopathic bacteria could be an important factor in the induction of apoptosis of host cells in the aetiology of periodontal diseases. PMID:10348360

  1. Scouring Potential of Mesophile Acidic Proteases of Pseudomonas aeruginosa for Grey Cotton Fabrics

    Saravanan, D.

    2013-04-01

    Mesophile, acidic proteases were produced using the microbial source, Pseudomonas aeruginosa, with wider thermal tolerances. Process conditions of scouring treatment were optimized using Taguchi method for optimum temperature, time, pH and concentration of protease. Treatment with the protease lower weight loss values compared to the alkali scouring, however, significant improvement in the absorbency compared to the grey samples was observed. Large amounts of pectin left out in the samples resulted in higher extractable impurities, substantiated by the FTIR results. Relatively, lower reduction in the tear strengths was observed in both warp and weft directions after protease treatment of the cotton fabrics.

  2. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production. PMID:27451238

  3. Proteases virais: importantes alvos terapêuticos de compostos peptideomiméticos

    Estela Maris Freitas Muri

    2014-04-01

    Full Text Available Proteases catalyze the hydrolysis of peptide bonds of proteins and peptides to produce smaller peptides and free amino acids. These enzymes are involved in physiologic processes such as blood coagulation and cellular death, and are related to life cycle of several viruses, such as hepatitis C, dengue, and AIDS. These features make most of proteases very important therapeutic targets for new pharmaceutical compounds. The development of peptidemimetics with improved pharmacokinetic properties is driving extensive research in the field of viral protease inhibitors. The present paper aims to highlight the design and synthesis of peptidemimetics that are able to inhibit viral proteases related to hepatitis C, dengue, and AIDS.

  4. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C. (WSU-MED); (NWU)

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  5. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  6. Synthesis and herbicidal evaluation of novel benzothiazole derivatives as potential inhibitors of D1 protease.

    Huang, Tonghui; Sun, Jie; An, Lin; Zhang, Lixian; Han, Cuiping

    2016-04-01

    D1 protease is a C-terminal processing protease that has been predicted to be an ideal herbicidal target. Three novel series of benzothiazole derivatives were synthesized and evaluated for their herbicidal activities against Brassica napus (rape) and Echinochloa crusgalli (barnyard grass). The preliminary bioassay indicated that most of the synthesized compounds possess promising D1 protease inhibitory activities and considerable herbicidal activities. Molecular docking was performed to position representative compounds into the active site of D1 protease to determine a probable binding model. PMID:26905829

  7. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  8. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  9. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16+ natural killer cells and CD3+, CD16- T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  10. Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

    Mulenga, A; Kim, T.; Ibelli, A. M. G.

    2013-01-01

    We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross-class inhibitor of both serine- and papain-like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics anal...

  11. Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide.

    Chen, Jue; Teixeira, Pedro Filipe; Glaser, Elzbieta; Levine, Rodney L

    2014-12-01

    The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP. PMID:25236746

  12. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis. PMID:26321757

  13. The Trypanosoma cruzi protease cruzain mediates immune evasion.

    Patricia S Doyle

    2011-09-01

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min and no increase in ∼P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.

  14. Comparison of HIV-1 protease expression in different fusion forms.

    Wan, M; Takagi, M; Loh, B N; Imanaka, T

    1995-06-01

    Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7663445

  15. A multifaceted analysis of HIV-1 protease multidrug resistance phenotypes

    Doherty Kathleen M

    2011-12-01

    Full Text Available Abstract Background Great strides have been made in the effective treatment of HIV-1 with the development of second-generation protease inhibitors (PIs that are effective against historically multi-PI-resistant HIV-1 variants. Nevertheless, mutation patterns that confer decreasing susceptibility to available PIs continue to arise within the population. Understanding the phenotypic and genotypic patterns responsible for multi-PI resistance is necessary for developing PIs that are active against clinically-relevant PI-resistant HIV-1 variants. Results In this work, we use globally optimal integer programming-based clustering techniques to elucidate multi-PI phenotypic resistance patterns using a data set of 398 HIV-1 protease sequences that have each been phenotyped for susceptibility toward the nine clinically-approved HIV-1 PIs. We validate the information content of the clusters by evaluating their ability to predict the level of decreased susceptibility to each of the available PIs using a cross validation procedure. We demonstrate the finding that as a result of phenotypic cross resistance, the considered clinical HIV-1 protease isolates are confined to ~6% or less of the clinically-relevant phenotypic space. Clustering and feature selection methods are used to find representative sequences and mutations for major resistance phenotypes to elucidate their genotypic signatures. We show that phenotypic similarity does not imply genotypic similarity, that different PI-resistance mutation patterns can give rise to HIV-1 isolates with similar phenotypic profiles. Conclusion Rather than characterizing HIV-1 susceptibility toward each PI individually, our study offers a unique perspective on the phenomenon of PI class resistance by uncovering major multidrug-resistant phenotypic patterns and their often diverse genotypic determinants, providing a methodology that can be applied to understand clinically-relevant phenotypic patterns to aid in the

  16. Effect of protease inhibitors (indinavir and ritonavir on the pharmacokinetics of gliclazide in rabbits

    Shaik Mastan

    2011-01-01

    Full Text Available Kilari Eswar Kumar1, Shaik Mastan2,31Pharmacology Division, University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India; 2Research and Development Cell, Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh, India; 3Cytel Statistical Software and Services Pvt Ltd, Pune, Maharashtra, IndiaAbstract: The objective of this study was to investigate the effect of protease inhibitors (indinavir and ritonavir on the pharmacokinetics of gliclazide in rabbits and to evaluate the mechanism of interaction of the combination. Studies in rabbits were conducted with oral doses of gliclazide, selected protease inhibitor, and their combination with a 1-week washout period between each treatment (single dose followed by multiple dose treatment. Blood samples were collected at regular time intervals by marginal ear vein puncture and serum gliclazide levels were analyzed by high-pressure liquid chromatography. Pharmacokinetic analysis was performed by noncompartmental analysis using WinNonlin Software. In combination, ritonavir significantly increased serum gliclazide levels and altered the pharmacokinetic parameters of gliclazide in rabbits while indinavir had no significant effect. The percentage increase of serum gliclazide level was 22.34% and 27.78% following single-dose and multiple-dose treatment of ritonavir, respectively. The interaction of ritonavir with gliclazide is pharmacokinetic at a metabolic level (by CYP3A4 inhibition in normal rabbits, while the interaction of indinavir with gliclazide is pharmacodynamic, which needs dose adjustment, and care should be taken when these combinations are prescribed for their clinical benefit in diabetic patients.Keywords: gliclazide, indinavir, ritonavir, diabetes, HIV infection, pharmacokinetics

  17. Prions in Variably Protease-Sensitive Prionopathy: An Update

    Laura Pirisinu; Jan Langeveld; Jue Yuan; Xiangzhu Xiao; Wen-Quan Zou; Pierluigi Gambetti

    2013-01-01

    Human prion diseases, including sporadic, familial, and acquired forms such as Creutzfeldt-Jakob disease (CJD), are caused by prions in which an abnormal prion protein (PrPSc) derived from its normal cellular isoform (PrPC) is the only known component. The recently-identified variably protease-sensitive prionopathy (VPSPr) is characterized not only by an atypical clinical phenotype and neuropathology but also by the deposition in the brain of a peculiar PrPSc. Like other forms of human prion ...

  18. Antiretroviral activity of protease inhibitors against Toxoplasma gondii

    Lianet Monzote

    2013-02-01

    Full Text Available The introduction of highly active antiretroviral therapy (HAART has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE. These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

  19. Expression and activation of proteases in co-cultures.

    Paduch, Roman; Kandefer-Szerszeń, Martyna

    2011-01-01

    The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested. Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells. In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the

  20. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease

    Vikram Surendra

    2010-07-01

    Full Text Available Abstract Background Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min. Results Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+ and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin. Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C. Conclusions An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

  1. Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus.

    Poorafshar, M; Aveskogh, M; Munday, B; Hellman, L

    2000-11-01

    To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases. PMID:11132153

  2. Study of the catalytic properties of bacillus subtilis proteases Estudio de las propiedades catalíticas de las proteasas bacillus subtilis

    Salcedo L.; Castellanos O.; Grebeshova R.

    1998-01-01

    The catalytic properties of proteases isolated from the filtrate of submerged fermentation of Bacillus subtilis were investigated. Proteases present in the filtrate were determined to be of the serine protease type based on the use of specific protease inhibitors; ethylenediamintetraacetic acid (EDTA) was used as a metalloprotease inhibitor, and phenylmethylsulfonylfluoride (PMSF) was used as a serine protease inhibitor. Protease activity was highly stable in alkaline solutions and at high te...

  3. A1C test

    HbA1C test; Glycated hemoglobin test; Glycosylated hemoglobin test; Hemoglobin glycosylated test; Glycohemoglobin test ... have recently eaten does not affect the A1C test, so you do not need to fast to ...

  4. Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

    Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

    2016-05-01

    Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. PMID:26849963

  5. Isolation, purification and characterization of extracellular protease produced by marine-derived endophytic fungus Xylaria psidii KT30

    Bugi Ratno Budiarto

    2015-01-01

    Full Text Available Objective: To isolate, purify and characterize extracellular protease produced by Xylaria psidii (X. psidii KT30. Methods: In the present study, the extracellular protease secreted by X. psidii KT30 was isolated and purified by using three steps of protein purification, then the purified protease was characterized by applying qualitative and quantitative enzymatic assays. Results: Extracellular protease with molecular mass 71 kDa has been purified successfully by applying diethylaminoethanol-Sepharose followed by sephadex SG75 with its final specific protease activity of 0.091 IU/mg. Protease was the most active at temperature 60 °C and pH 7. The activity of enzyme was abolished mostly by phenylmethanesulfonyl fluoride, showing it is family of serine protease. Conclusions: Extracellular serine protease produced by X. psidii KT30 with good biochemical properties displayed some promising results for its further application in field of biotechnology or medicine.

  6. Evidence of a new serine protease in the rat pure pancreatic juice that degrades somatostatin

    Somatostatin (SS) is found in the endocrine pancreas and has been reported in the pure pancreatic juice (PPJ) of different species. Characterization by gel filtration of immunoreactive SS (irSS) in the rat PPJ (rPPJ) results in a single peak corresponding to 23kDa molecular weight. Incubation of the 23kDa fraction with labeled or synthetic SS results in time dependent degradation of both peptides. This degradation is inhibitable by PMSF, calcium and by heat, whereas specific inhibitors of trypsin and chymotrypsin are without effect. These data suggest that irSS previously measured in rPPJ samples by RIA without confirmation of radioactive tracer stability may lead to false positive results. Indeed, our study indicates the presence of a 23kDa enzyme in the rPPJ degrading radiolabeled somatostatin during the RIA procedure. This putative new enzyme found into the rPPJ may thus be partially responsible for the apparent irSS presence

  7. Protease activation involved in resistance of human cells to x-ray cell killing

    Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

  8. Imbalance between Cysteine Proteases and Inhibitors in a Baboon Model of Bronchopulmonary Dysplasia

    Altiok, Ozden; Yasumatsu, Ryuji; Bingol-Karakoc, Gulbin; Riese, Richard J.; Stahlman, Mildred T.; Dwyer, William; Pierce, Richard A.; Bromme, Dieter; Weber, Ekkehard; Cataltepe, Sule

    2005-01-01

    Rationale: Bronchopulmonary dysplasia (BPD) continues to be a major morbidity in preterm infants. The lung pathology in BPD is characterized by impaired alveolar and capillary development. An imbalance between proteases and protease inhibitors in association with changes in lung elastic fibers has been implicated in the pathogenesis of BPD.

  9. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N

    2016-09-01

    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity. PMID:27278806

  10. 21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

    2010-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bacterially-derived protease enzyme preparation... enzyme preparation. (a) Bacterially-derived protease enzyme preparation is obtained from the...

  11. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product.

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Mixed carbohydrase and protease enzyme product. 184... enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes... current good manufacturing practice conditions of use: (1) The ingredient is used as an enzyme, as...

  12. Influence of food preservatives, heat, and gamma radiation on protease production by aeromonas hydrophila

    The effect of food preservatives, heat and gamma radiation on growth and protease production by A. Hydrophila were studied. Growth of A. hydrophila was inhibited at 5% sodium chloride, whereas protease activity was not detected at 4% sodium chloride. Sodium citrate, sodium acetate, potassium sorbate, and sodium nitrite affected growth and production at Ph 6.0 Numbers of viable cells and protease production by A. hydrophila in broth medium and in minced meat slurries decreased by increasing the time of heating in a water bath set at 50 degree C while they were rapidly reduced and no detection of protease after heating for 2 min. At 55 degree C or 60 degree C. The activity of protease was found to be stable at 60 degree C for 30 min. and the enzyme activity decreased by 50% at 80 degree C for 5 min. and retained stable at 100 degree C or 121 degree C for 5 min. indicating the thermal resistance of protease. gamma radiation at dose level of 1.5 kGy inactivated the growth and protease production. Food preservatives, heat, and gamma radiation are required to control the growth of A. hydrophila and protease production in foods

  13. Inhibition of viral proteases by Zingiberaceae extracts and flavones isolated from Kaempferia parviflora.

    Sookkongwaree, K; Geitmann, M; Roengsumran, S; Petsom, A; Danielson, U H

    2006-08-01

    In order to identify novel lead compounds with antiviral effect, methanol and aqueous extracts of eight medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). In general, the methanol extracts inhibited the enzymes more effectively than the aqueous extracts. HIV-1 protease was strongly inhibited by the methanol extract of Alpinia galanga. This extract also inhibited HCV and HCMV proteases, but to a lower degree. HCV protease was most efficiently inhibited by the extracts from Zingiber officinale, with little difference between the aqueous and the methanol extracts. Many of the methanol extracts inhibited HCMV protease, but the aqueous extracts showed weak inhibition. In a first endeavor to identify the active constituents, eight flavones were isolated from the black rhizomes of Kaempferia parviflora. The most effective inhibitors, 5-hydroxy-7-methoxyflavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease with IC50 values of 19 microM. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease with IC50 values of 190 and 250 microM, respectively. PMID:16964717

  14. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis

    Kádek, Alan; Tretyachenko, V.; Mrázek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, M.; Schriemer, D. C.; Man, Petr

    2014-01-01

    Roč. 95, MAR 2014 (2014), s. 121-128. ISSN 1046-5928 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : Plant aspartic protease * Nepenthesin * Protease characterization Subject RIV: CE - Biochemistry Impact factor: 1.695, year: 2014

  15. Kinetics of the dimerization of retroviral proteases: The "fireman's grip" and dimerization

    Ingr, Marek; Kondrová, Taťána; Stříšovský, Kvido; Majerová, E.; Konvalinka, Jan

    2003-01-01

    Roč. 12, - (2003), s. 2173-2182. ISSN 0961-8368 R&D Projects: GA MZd NI6339 Institutional research plan: CEZ:AV0Z4055905 Keywords : retroviral protease * dimerization * HIV protease Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2003

  16. Detection of cysteine protease in Taenia solium-induced brain granulomas in naturally infected pigs

    Mkupasi, Ernatus Martin; Sikasunge, Chummy Sikalizyo; Ngowi, Helena Aminiel;

    2013-01-01

    In order to further characterize the immune response around the viable or degenerating Taenia solium cysts in the pig brain, the involvement of cysteine protease in the immune evasion was assessed. Brain tissues from 30 adult pigs naturally infected with T. solium cysticercosis were subjected...... protease may play a role in inducing immune evasion through apoptosis around viable T. solium cysts....

  17. Identification and characterization of alkaline serine protease from goat skin surface metagenome.

    Pushpam, Paul Lavanya; Rajesh, Thangamani; Gunasekaran, Paramasamy

    2011-01-01

    Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively. PMID:21906326

  18. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  19. Full quantum mechanical study of binding of HIV-1 protease drugs

    Zhang, Da W.; Zhang, John Z. H.

    Fully quantum mechanical studies of detailed binding interactions between HIV-1 protease and six FDA (Food and Drug Administration)-approved drugs (saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir) are carried out using a recently developed MFCC (molecular fractionation with conjugate caps) method. The MFCC calculation produces a quantum mechanical interaction spectrum for any protease drug binding complex. Detailed quantitative analysis on binding of lopinavir to specific residues of the protease is given from the current study. The present calculation shows that the dominant binding of lopinavir to the protease is through the formation of a strong hydrogen bond between the central hydroxyl group of the drug to the aspartate oxygen of Asp25 in one of the two chains of the protease (A chain). This is closely followed by hydrogen binding of the drug to Asp29 in the B chain and somewhat weak hydrogen bonding to Asp30, Gly27, Gly48, and Ile50 in both chains. By partitioning all six drugs into four building blocks besides the central component containing the hydroxyl group, MFCC calculation finds that block III has essentially no binding interaction with the protease and the major binding interactions of these drugs are from blocks II and IV, in addition to the dominant central hydroxyl group. This detailed quantitative information on drug binding to the protease is very useful in rational design of new and improved inhibitors of HIV-1 protease and its mutants.

  20. Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors

    Bayés, A.; Comellas-Bigler, M.; Rodriquez de la Vega, M.; Maskos, K.; Bode, W.; Aviles, F.X.; Beekwilder, M.J.; Vendrell, J.

    2005-01-01

    Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptid

  1. Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors

    Pehere, A.D.; Pietsch, M.; Gütschow, M.; Neilsen, P.M.; Pedersen, Daniel Sejer; Nguyen, S.; Zvarec, O.; Sykes, M.J.; Callen, D.F.; Abell, A. D.

    2013-01-01

    Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole- containing macrocyclic protease inhibitors pre-organized into a b-strand conformation and an evaluation of their...

  2. The optimization of fermentation conditions and enzyme properties of Stenotrophomonas maltophilia for protease production.

    Wang, Zaigui; Sun, Linghong; Cheng, Jia; Liu, Chaoliang; Tang, Xiangfang; Zhang, Hongfu; Liu, Ying

    2016-03-01

    Intestinal bacteria play a significant physiological role in silkworms. Proteases secreted by intestinal microbes can promote the digestion of the nutrient by Bombyx mori and the absorption of mulberry leaves. Intestinal bacteria from Jingsong × Haoyue in the fourth larvae were isolated and purified to obtain high activity protease-producing bacteria. The morphology of the identified bacterial colony was examined by microscopy combined with the 16S rDNA method. The results showed that this bacterium was Gram negative and that it belonged to Stenotrophomonas maltophilia, which produces the proteases. To improve the utilization rate of these proteases, we studied the proper culture conditions for producing proteases, and we further studied the properties of the proteases that were produced. The results showed that the optimal enzyme-producing conditions were as follows: pH of 7.0, culture temperature of 35 °C, incubation time of 36 H, and outfit fluid amount of 60 mL per 100 mL. Meanwhile, the properties of the preliminary enzyme purification indicated that the best pH of the enzymes was 9.0 and the optimal reaction temperature was 50 °C. The enzymes are alkaline proteases that show satisfactory stability at 30 °C and pH 9.0. Consequently, it is suitable for the proteases secreted by S. maltophilia to play a bioactive role in the silkworm gut. PMID:25656812

  3. Feeding Anthrax: The Crystal Structure of Bacillus anthracis InhA Protease.

    Schacherl, Magdalena; Baumann, Ulrich

    2016-01-01

    Pathogenic bacteria secrete proteases to evade host defense and to acquire nutrients. In this issue of Structure, Arolas et al. (2016) describe the structural basis of activation and latency of InhA, a major secreted protease of Bacillus anthracis. PMID:26745525

  4. Isolation, identification and optimization of alkaline protease production by Candida viswanathii

    Mandana Lotfi

    2014-03-01

    Conclusion: Due to the high demand for industrial enzymes in the Country and the high activity of alkaline proteases produced by strain. It seems that the native strain can achieve high production of alkaline proteases.These native strains could be resulted in the independence of our country in industrial enzymes production.

  5. Crystal Structure of a Novel Viral Protease with a Serine/Lysine Catalytic Dyad Mechanism

    Feldman,A.; Lee, J.; Delmas, B.; Paetzel, M.

    2006-01-01

    The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.

  6. Hunting-ton for New Proteases: MMPs as the New Target?

    Johri, Ashu; Beal, M. Flint

    2010-01-01

    Mutant huntingtin proteolysis mediated by various proteases plays a key role in Huntington's disease (HD) pathogenesis. In this issue of Neuron, Miller et al. have identified 11 proteases, including matrix metalloproteinases (MMPs), that when inhibited reduce huntingtin proteolysis and produce beneficial therapeutic effects. These findings provide new insights into huntingtin proteolysis and its potential as a therapeutic target.

  7. Conditions for the modification of radiation transformation in vitro by a tumor promoter and protease inhibitors

    These experiments were designed to define the conditions necessary for the modification of radiation-induced transformation in C3H/10T1/2 cells by TPA and protease inhibitors. The results show that: (i) the lowest effective dose of various protease inhibitors to suppress transformation in vitro varies over several orders of magnitude; on a molar basis, the inhibitors of chymotrypsin appear to be the most effective protease inhibitors at suppression of radiation-induced transformation in vitro, (ii) the protease inhibitors antipain and the Bowman-Birk (soybean) protease inhibitor have no effect on radiation transformation when present only during irradiation, (iii) the protease inhibitor antipain can suppress radiation transformation in vitro when applied to proliferating initiated cells as late as 10 days and 13 cell divisions post-irradiation, and (iv) TPA treatment following a 10-day protease inhibitor (anti-pain) exposure of X-irradiated initiated cells does not lead to promotion in vitro. These results suggest that protease inhibitor treatment of the initiated cells has irreversibly reverted cells to their original or uninitiated condition which existed before irradiation

  8. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

    Studdert, C A; Herrera Seitz, M K; Plasencia, I; Sanchez, J J; de Castro, R E

    2001-01-01

    other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests that...

  9. Short hydrogen bonds in the catalytic mechanism of serine proteases

    VLADIMIR LESKOVAC

    2008-04-01

    Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

  10. Cysteine protease activation and apoptosis in Murine norovirus infection

    Ettayebi Khalil

    2009-09-01

    Full Text Available Abstract Background Noroviruses are the leading cause of viral gastroenteritis. Because a suitable in vitro culture system for the human virus has yet to be developed, many basic details of the infection process are unknown. Murine norovirus (MNV serves as a model system for the study of norovirus infection. Recently it was shown that infection of RAW 264.7 cells involved a novel apoptotic pathway involving survivin. Results Using a different set of approaches, the up-regulation of caspases, DNA condensation/fragmentation, and membrane blebbing, all of which are markers of apoptosis, were confirmed. Live cell imaging and activity-based protein profiling showed that activation of caspase-like proteases occurred within two hours of infection, followed by morphological changes to the cells. MNV infection in the presence of caspase inhibitors proceeded via a distinct pathway of rapid cellular necrosis and reduced viral production. Affinity purification of activity-based protein profiling targets and identification by peptide mass fingerprinting showed that the cysteine protease cathepsin B was activated early in infection, establishing this protein as an upstream activator of the intrinsic apoptotic pathway. Conclusion This work adds cathepsin B to the noncanonical programmed cell death induced by MNV, and provides data suggesting that the virus may induce apoptosis to expand the window of time for viral replication. This work also highlights the significant power of activity-based protein profiling in the study of viral pathogenesis.

  11. Moringa oleifera Lam.: Protease activity against blood coagulation cascade

    A Satish

    2012-01-01

    Full Text Available Background : The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae leaf (MOL and root (MOR. Materials and Methods : Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Results : Caseinolytic activity of MOL was significantly higher (P ≤ 0.05 than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05 plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05 in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen and fibrinolytic activity (human plasma clot was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aa and Bb subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. Conclusions : These findings support the traditional usage of M. oleifera extracts for wound healing.

  12. Design of HIV Protease Inhibitors Based on Inorganic Polyhedral Metallacarboranes

    Rezacova, Pavlina; Pokorna, Jana; Brynda, Ji; Kozisek, Milan; Cigler, Petr; Lesik, Martin; Fanfrlik, Jindrich; Rezac, Jan; Saskova, Klara Grantz; Sieglova, Irena; Plesek, Jaromir; Sicha, Vaclav; Gruner, Bohumir; Oberwinkler, Heike; Sedlacek, Juraj; Krausslich, Hans-Georg; Hobza, Pavel; Kral, Vladimir; Konvalinka, Jan; (Heidelberg); (ASCR-ICP); (ICT-Czech)

    2010-04-19

    HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H{sub 2}N-(8-(C{sub 2}H{sub 4}O){sub 2}-1,2-C{sub 2}B{sub 9}H{sub 10})(1',2'-C{sub 2}B{sub 9}H{sub 11})-3,3'-Co){sub 2}]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

  13. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  14. Purification and characterization of alkaline protease from Lysinibacillus fusiformis

    Suppiah S*

    2012-08-01

    Full Text Available A novel alkaline protease producing bacterium was isolated from the gut of an estuarine fish Etroplus suratensis. The strain was identified by sequencing the fragment of their bacterial 16s rRNA and its homology was 97% closest to the Lysinibacillus fusiformis. An extracellular protease from this organism was purified by acetone precipitation, ion exchange chromatography and gel filtration chromatography methods and the specific activity of the purified enzyme was found to be 20.39 U/mg, 169.46U/mg and 352.0U/mg respectively. The molecular weight of the purified enzyme was determined to be 29kDa through SDS/PAGE analysis. The enzyme showed that the maximum at pH 9.0 and temperature at 40ºC. The purified enzyme remains active in the presence of various metal ions and it was strongly stimulated by the addition of Ca2+. Among the tested surfactants, the optimum activity was observed in SDS when compared to the other tested surfactants. Normal 0 false false false EN-US X-NONE X-NONE

  15. Development of an Aeromonas hydrophila recombinant extracellular protease vaccine.

    Wu, Lei; Jiang, Ya-nan; Tang, Qian; Lin, Hui-xing; Lu, Cheng-ping; Yao, Huo-chun

    2012-01-01

    Aeromonas hydrophila (Ah) exists widely in the aquatic environment and infects a variety of animals. Extracellular protease (EPR) is an important protective antigen that induces a specific antibody response to resist Ah infection. In this study, two genes encoding extracellular protease epr2 and epr3 were linked within the expression vector pET32a to construct a recombinant pET-epr2-3 plasmid. The immunogenicity of the fusion protein epr2-3 was investigated as a subunit vaccine in ICR mice. The recombinant epr2-3 protein induced the production of high antibody titers. The survival rate against homogenous Ah J-1 challenge was significantly higher in the epr2-3 vaccinated group (≥80%) compared with the inactivated Ah vaccinated group and the challenge control group (P < 0.01), thus indicating that the recombinant epr2-3 protein provided significant protection against Ah infection. Therefore, the recombinant epr2-3 is a promising candidate for development as a vaccine against Ah infections. PMID:22874879

  16. Protease activation in glycerol-based deep eutectic solvents

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2011-01-01

    Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmo l min−1 g−1) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally. PMID:21909232

  17. ADAMTS: a novel family of extracellular matrix proteases.

    Tang, B L

    2001-01-01

    ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling. PMID:11167130

  18. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (Mpro), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV Mpro was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6122. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function

  19. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex w...

  20. Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4.

    Wang, X C; Zhao, H Y; Liu, G; Cheng, X J; Feng, H

    2016-01-01

    The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries. PMID:27323184

  1. Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target

    Sundararaj, Srinivasan; Saxena, Ajay K.; Sharma, Ruby; Vashisht, Kapil; Sharma, Supriya; Anvikar, Anup; Dixit, Rajnikant; Rosenthal, Philip J.; Pandey, Kailash C.

    2014-01-01

    Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) a...

  2. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    Raheem Ullah; Majid Ali Shah; Soban Tufail; Fouzia Ismat; Muhammad Imran; Mazhar Iqbal; Osman Mirza; Moazur Rhaman

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally ...

  3. Interaction Characteristics of Viral Protease Targets and Inhibitors : Perspectives for drug discovery and development of model systems

    Shuman, Cynthia F

    2003-01-01

    Viral proteases are important targets for anti-viral drugs. Discovery of protease inhibitors as anti-viral drugs is aided by an understanding of the interactions between viral protease and inhibitors. This thesis addresses the characterization of protease-inhibitor interactions for application to drug discovery and model system development. The choice of a relevant target is essential to molecular interaction studies. Therefore, full-length NS3 protein of hepatitis C virus (HCV) was obtained,...

  4. Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

    Hayama, Tomomi; Kamio, Naoto; Okabe, Tatsu; Muromachi, Koichiro; Matsushima, Kiyoshi

    2016-07-01

    Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566265

  5. Phenylalanine and Phenylglycine Analogues as Arginine Mimetics in Dengue Protease Inhibitors.

    Weigel, Lena F; Nitsche, Christoph; Graf, Dominik; Bartenschlager, Ralf; Klein, Christian D

    2015-10-01

    Dengue virus is an increasingly global pathogen. One of the promising targets for antiviral drug discovery against dengue and related flaviviruses such as West Nile virus is the viral serine protease NS2B-NS3. We here report the synthesis and in vitro characterization of potent peptidic inhibitors of dengue virus protease that incorporate phenylalanine and phenylglycine derivatives as arginine-mimicking groups with modulated basicity. The most promising compounds were (4-amidino)-L-phenylalanine-containing inhibitors, which reached nanomolar affinities against dengue virus protease. The type and position of the substituents on the phenylglycine and phenylalanine side chains has a significant effect on the inhibitory activity against dengue virus protease and selectivity against other proteases. In addition, the non-natural, basic amino acids described here may have relevance for the development of other peptidic and peptidomimetic drugs such as inhibitors of the blood clotting cascade. PMID:26367391

  6. Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

    2008-01-01

    The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .

  7. Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

    2008-01-01

    The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.

  8. Lipases and proteases produced by indigenous Pseudomonas aeruginosa strain as potential detergent additives

    Grbavčić Sanja Ž.

    2009-01-01

    Full Text Available Enzymes produced by indigenous Pseudomonas aeruginosa strain have been subjected to research considering their potential application as detergent additives. As previously noted, lipase produced by Pseudomonas aeruginosa is highly alkaline, thermostable and solvent tolerant. Furthermore, same strain exhibits both lipase and protease activity establishing this lipase as potentially desirable component of enzyme-containing detergents. Further research was carried out to investigate insusceptibility of this lipase against coexisting native protease, several commercial surfactants, oxidizing agents and commercial detergents. Lipases and proteases remained highly active when incubated with several different surfactants and oxidizing agents under washing conditions. Moreover, presence of surfactants and oxidizing agents such as Tween® 20 and Triton® X-100 initially augment lipase and protease activity. Additionally, crude lipase preparation was insusceptible to coexisting native protease hence indicating possible storage stability. Overall, the remarkable properties of these enzymes make them potential detergent additives.

  9. ‘Seeding’ with protease to optimize protein crystallization conditions in in situ proteolysis

    Addition of protease instead of seeds using a robot can be used to optimize the concentration of protease in in situ proteolysis experiments and has been successfully tested using two proteins. In situ proteolysis is one of the most effective rescue strategies for protein crystallization, and optimization of the ratio between the protein and the protease is one of the key steps in the process. Seeding is a very powerful tool to optimize crystallization conditions and can be performed by most crystallization robots. Addition of protease instead of seed stock using a robot can be used to optimize the concentration of protease in in situ proteolysis experiments and has been successfully tested using two proteins

  10. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

    Pieper Rembert

    2011-05-01

    Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

  11. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    Sissons, James; Alsam, Selwa; Goldsworthy, Graham; Lightfoot, Mary; Jarroll, Edward L; Khan, Naveed Ahmed

    2006-01-01

    Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae. PMID:16672059

  12. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    Jarroll Edward L

    2006-05-01

    Full Text Available Abstract Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM, which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM, elastin (elastic fibrils of ECM, plasminogen (involved in proteolytic degradation of ECM, as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC. Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s in invasion of the brain tissue by amoebae.

  13. Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

    Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L.; Schiffer, Celia A. (CIT); (UMASS, MED)

    2012-10-23

    HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

  14. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry. PMID:25224381

  15. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu;

    2016-01-01

    -ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its CDR-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to...

  16. Increased proportions of bacteria capable of cleaving IgA1 in the pharynx of infants with atopic disease

    Kilian, M; Husby, S; Høst, A;

    1995-01-01

    Neisseria meningitidis, of which the first mentioned species was mainly responsible for the difference observed at the 18-mo examination. Percentage proportions of IgA1 protease-producing bacteria were significantly related to passive smoking which may stimulate the premature and more pronounced pharyngeal...

  17. Protease exógena em dietas para juvenis de tucunaré-paca (Cichla sp. Exogenous protease in diets for tucunaré paca (Cichla sp. juvenile

    Emerson Carlos Soares

    2008-06-01

    Full Text Available Neste experimento foi analisada a atividade enzimática da protease exógena nos tratos digestórios dos peixes e, posteriormente, o desempenho zootécnico dos juvenis de tucunaré-paca (Cichla sp. por intermédio da inclusão de três níveis de protease exógena na dieta com 40% proteína bruta mais a dieta controle. Para avaliar o melhor nível de inclusão de enzimas, foram elaboradas quatro dietas em forma de peletes com quatro níveis de inclusão de protease exógena (0,00; 0,05; 0,10 e 0,15%, em um delineamento inteiramente casualisado (fatorial 4 × 4. Os melhores resultados de ganho de peso, conversão alimentar e crescimento específico em peso foram apresentados pelos animais alimentados com a dieta contendo 0,10% de protease exógena. Concluiu-se que a enzima protease exógena adicionada à dieta influenciou os parâmetros corpóreos dos juvenis de tucunaré-paca.This experiment analyzed the enzymatic activity of the exogenous protease in the fish digestive tract and, afterwards, it was also evaluated the growth performance of tucunaré paca (Cichla sp. juvenile by the addition of four exogenous protease levels to a control diet with 40% CP. To evaluate the best enzyme inclusion level, four pelleted diets were elaborated with the following exogenous protease levels (0.00, 0.05, 0.10 and 0.15%, in a completely randomized design in a 4 × 4 factorial arrangement. The best results of average weight gain, feed conversion ratio, and weight specific growth rate were showed by the animals fed with diet containing 0.10% of exogenous protease. The exogenous protease enzyme added to the diet affected the body characteristics of tucunaré paca juvenile.

  18. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

    Zongyun Chen

    Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

  19. Cathepsin B protease is required for metamorphism in silkworm,Bombyx mori

    Gen-Hong Wang; Chun Liu; Qing-You Xia; Xing-Fu Zha; Jie Chen; Liang Jiang

    2008-01-01

    Cathepsin B belongs to lysosomal cysteine protease of the papain family.Temporal and spatial expression analysis of cathepsin B of Bombyx mori (BmCtB) was carried out based on Expression Sequence Tags (ESTs) data,oligonucleotide microarray,reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR.Expression of BmCtB was observed in all of the tissues and stages.Among the 10 tested tissues,the fat body and posterior silk gland are the two most enriched tissues with BmCtB.During Bombyx development,there was an expression fastigium of BmCtB during metamorphosis.RNA interference was used to suppress the expression of cathepsin B during metamorphosis.Significant developmental defective phenotypes were obtained in the RNAi treated group.The dramatically reduced expression of BmCtB was confirmed by Northern blot and quantitative real-time PCR.These evidences strongly suggest cathepsin B proteinase was predominantly involved in the metabolism process of fat body and the posterior silk gland and was critical for metamorphism and development of silkworm,Bombyx mori.

  20. A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii

    Menyhárd, Dóra K.; Kiss-Szemán, Anna; Tichy-Rács, Éva; Hornung, Balázs; Rádi, Krisztina; Szeltner, Zoltán; Domokos, Klarissza; Szamosi, Ilona; Náray-Szabó, Gábor; Polgár, László; Harmat, Veronika

    2013-01-01

    Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025